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P. fl i ; as l J& “xiv-P6534 ‘. -p 5. 4" 5"” i U??? , - ~ . l. s: .7 ~' "I: D \ This is to certify that the dissertation entitled The Enzymatic Mechanism of Ribulose Bisphosphate Carboxylase/Oxygenase presented by Robert Michael Mulligan has been accepted towards fulfillment of the requirements for Ph.D. degteni Biochemistny new Major professor [hue June 28, 1983 MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 remove this checkout from your record. FINES will ———7 be charged if book is returned after the date stamped below. MSUA gEjURNING MATERIALS: Place in book drop to LIBRARIES ”- ' "" "" ~" :. :~ . a.- .- a...” a 4 Q ‘2 '- . . ll 4 _ 1g 3: It .6 & Lee- a has... 3. sun,- 1:...2 a u '-. i MAR,0_42002 " SW ”SE 0.? THE ENZYMATIC MECHANISM OF RIBULOSE BISPHOSPHATE CARBOXYLASE/OXYGENASE BY Robert Michael Mulligan A DISSERTATION submitted to Michigan State University in partial fulfillment of the requirement for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1983 Has 3!“? ABSTRACT THE ENZYMATIC MECHANISM OF RIBULOSE BISPHOSPHATE CARBOXYLASE/OXYGENASE BY Robert Michael Mulligan The mechanism of ribulose bisphosphate carboxylase/oxygenase has been examined. The carboxylated intermediate was liberated from a carboxylase reaction by acidification and stabilized by reduction. The reduction product was identified as carboxy- arabinitol bisphosphate and was obtained at a stoichiometry of 0.04 per enzyme active site. An intermediate with a labile phosphate ester was also liberated by acidification of a carboxylase reaction. Phosphate release occurred with a stoichiometry of about 0.04 per enzyme active site, and was probably due to the lability of the carboxylated intermediate. Attempts to demonstrate the presence of other intermediates of the carboxylase and oxygenase reactions failed to verify the ex- istence of these other intermediates. Glyoxylate is a slowly reversible inhibitor of ribulose bisphosphate carboxylase. Glyoxylate forms an adduct with a catalytic site amino acid residue which maybe involved in catalysis. Glyoxylate did not selectively inhibit either Robert Michael Mulligan carboxylase or oxygenase activities. Inhibition of carboxylase activity by glyoxylate exhibited an apparent dissociation constant of 3.3 mM and a maximal psuedo first order rate constant for inhibition of 7 X 10-3 second-1. The dissociation of glyoxylate from the enzyme exhibited a first order rate constant of 12 X 10-3 second-1. Glyoxylate also elicted a two-fold stimulation in carboxylase activity of the unactivated enzyme. The stimulation of activity by glyoxylate was not due to in- creased activation of the enzyme due to carbamate formation. Glyoxylate inhibited photosynthesis by intact chloroplasts, but, carboxylase activity was not substantially inhibited. Other weak acids also inhibited CO2 fixation, and the inhibition of- photosynthesis may be a general effect of weak acids. Glyoxylate was reduced to glycolate by illuminated chloroplasts and glyoxylate reductase may serve to scavenge and detoxify glyoxylate in chloroplasts. Digestion of activated spinach carboxylase with trypsin rapidly inhibits catalytic activity, but carboxyarabinitol bisphosphate binding is only slightly diminished. Gel electro- phoresis of the digested protein demonstrated that limited tryptic digestion of activated carboxylase converted the native large subunit (52,800 daltons) to 2 forms with molecular weights of 51,300 and 49,300 daltons. Thus carboxyarabinitol bisphos- phate binding may not be used to determine the catalytically competent active sites if proteolysis is a factor. ACKNOWLEDGEMENTS I would like to express my appreciation for financial support and professional guidance provided by Dr. N.E. Tolbert. Gratitude is also extended to Dr. K. Schubert who was involved in securing and maintaining the Jessie Smith Noyes Fellowship which provided financial ~support for 2 years. The research presented in this thesis is partially the result of collaborative efforts with other investigators. Dr. Cathy Cook was a co-investigator in the studies pre- sented in Chapter 3. Barbara Wilson was involved in the studies using intact chloroplasts in Chapter 4. Dr. Irwin A. Rose had considerable influence on the experiments pre- sented in Chapters 1 and 2. Dr. Ming Tien provided helpful discussions on oxygen chemistry. Dr. John Pierce contributed to many stimulating discussions. The members of my guidance committee have been very helpful. It has been a pleasure to have ready access to these individuals for scientific discussions. I would like to thank my parents for their love and patience. Their input into my personal and intellectual development is greatly appreciated. Thanks are also ex- tended to Mr. William Kushner and Dr. Christopher Papasian for stimulating my interest in science. Finally, the love and emotional support of my wife, Maureen Ford Mulligan, has ii carried me through the frustrating hours and shared with me the joyous moments. iii LIST OF TABLES TABLE OF CONTENTS LIST OF FIGURES LIST OF ABBREVIATIONS INTRODUCTION Chapter 1. Chapter 2. Synthesis and Assembly of Ribulose-P2 Carboxylase/ Oxygenase The Structure of the Catalytic Site The Reaction Mechanism of Ribulose-P Car- boxylase/angenase The Role of Ribulose-P Carboxylase/Oxygenasg in Photo- synthetic Carbon Metabolism THE LABILITY OF AN INTERMEDIATE OF THE RIBULOSE-PZCARBOXYLASE REACTION Materials and Methods Results Phosphate Lability of an Enzyme Liberated Intermediate Absence of Paracatalytic Inactivation of Ribulose-P2 Carboxylase/Oxygenase Paracatalytic Inactivation of Fructose-P2 Aldolase Discussion INVESTIGATIONS ON THE MECHANISM OF RIBULOSE-PZ OXYGENASE Materials and Methods Results The Presence and Stoichiometry of Flavin in Ribulose-P Carboxylase/Oxygenase Attempts to Observe Inter- iv Page vii viii 13 19 20. 24 24 35 36 37 42 43 43 43 53 mediates in the Ribulose-P2 Oxygenase Reaction Discussion Chapter 3. STIMULATION AND INHIBITION OF RIBULOSE-P CARBOXYLASE/OXYGENASE BY GLYOXYL TE Materials and Methods Results Inhibition of Ribulose-P Carboxy- lase/Oxygenase by Glyoiylate The Stimulation of the Unactivated Enzyme by Glyoxylate Discussion Chapter 4. THE EFFECT OF GLYOXYLATE ON PHOTO- SYNTHESIS BY INTACT CHLOROPLASTS ' Materials and Methods Chloroplasts Photosynthetic CO2 Fixation Assay of Ribulose-P Carboxylase from Intact Chlorgplasts Ribulose-P2 Determination Glyoxylate Reductase (NADP+) Paper Chromatographic Separation of Metabolites Fructose-P2 Determination Materials Results Activity and Activation of Ribulose- P Carboxylase in IRtact Chloroplasts Glyoxylate Metabolism in Chloroplasts The Effect of Salts of Weak Acids on Photosynthesis Effect of Glyoxylate Inhibition on the Products of Photosynthetic CO2 Fixation Discussion Chapter 5. THE EFFECT OF TRYPSIN ON RIBULOSE-P CARBOXYLASE/OXYGENASE 2 54 62 63 64 64 75 82 87 88 88 88 89 89 90 90 90 91 91 91 99 100 103 103' 108 Material and Methods Results Discussion Chapter 6. NONENZYMATIC OXIDATION OF RIBULOSE-P IN THE PRESENCE OF CYANIDE:AN ARTIFACT Materials and Methods Results Sugar-P Dependent O2 Consumption Intermediates of 0 Reduction EPR Spin Trapping Studies Discussion Chapter 7. THE EFFECT OF SOME POTENTIALLY REGULATORY COMPOUNDS ON RIBULOSE-P2 CARBOXYLASE/OXYGENASE Materials and Methods Results and Discussion The Effect of Fructose-P on Ribulose-P2 Carboxylasg/ Oxygenase The Effect of Trans-Z-hexenal on Ribulose-P2 Carboxylase/ Oxygenase The Effect of Ribose-S-P on Ribulose-P2 Carboxylase/ Oxygenase List of Publications Bibliography vi 111 114 127 130 130 132 132 132 145 150 152 152 153 153 157 160 164 166 LIST 9; TABLES Table l. The Stability of the Phosphate Labile Intermediate. 2. Binding of CO , CABP and Glyoxylatg by Partially Activated Ribulose-P2 Carboxylase. 3. Summary of Kinetic Parameters of Ribulose-P Carboxylase from Various ources. 4. The Effect of Fructose-2,6-P on Ribulose-P2 Carboxylase/Ofiygenase. vii Page 29 81 115 154 LIST 9: FIGURES FIGURE 1. 10. 11. 12. 13. 14. Phosphate Liberation as a Function of the Amount of Ribulose-P2 Carboxylase Active Sites. Resolution of the Carboxylated Intermediate from the Ribulose-P Carboxylase Reaction after Acid Denaturagion and Reduction. Inhibition of Ribulose-P Carboxylase by the NaBH Reduction Pfoduct of the Carboxylated Intermediate. The Fluorescence Spectra of Native Ribulose-P2 Carboxylase/Oxygenase. The Fluorescence Spectra of Denatured Ribulose-P Carboxylase/Oxygenase. 2 The Fluorescence Spectra of Flavin Mononucleotide. Quantitation of Riboflavin by Fluorescence of Ribulose-P2 Carboxylase. Time Course of Inhibition of Ribulose-P Carboxylase Activity by Glyoxylate undef Various Conditions. - Inhibition of Ribulose-P Carboxylase by Glyoxylatg. The Inhibition of Carboxylase and Oxygenase Activities as a Function of Glyoxylate Concentration. The Reversibility of Glyoxylate Inhibition. The Stimulation of Ribulose-P2 Carboxylase Activity by Glyoxylate. The Effect of Gluconate-G-P and-Glyoxylate on Unactivated Ribulose-P2 Carboxylase. The Effect of Glyoxylate on Ribulose-P Carboxylase Activity in Chloroplasts. 2 viii PAGE 27 32 34 45 47 49 52 66 69 72 74 77 80 94 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. The Effect of Preincubation with Glyoxylate and Illumination on Ribulose-P2 Carboxylase Activity in Chloroplasts. The Effect of Glyoxylate on Ribulose-P Levels in Chloroplasts. 2 The Effect of Glyoxylate and Glycolate on Photosynthesis by Intact Chloroplasts. The Effect of Trypsin on Activity and CABP Binding by Ribulose-P2 Carboxylase. Dissociation of CABP from the Activated Enzyme-CABP Complex after Trypsin Digestion. SDS-gel Electrophoresis of Ribulose-P Carboxylase after Digestion by Trypsifi. Molecular Sieve Chromatography of Ribulose-Pz Carboxylase after Digestion with Trypsin. Dioxygen Consumption by Dihydroxyacetone-P in the Presence of Cyanide. Initial Rate of Dihydroxyacetone-P Dependent 0 Consumption as a Function of Cyanide Cancentration. Effect of pH on the Initial Rate of 0 Consumption with Ribulose-P2 and Cyanfde. Inhibition of the Initial Rate of O _ Consumption by the Manganese Superoiide Dismutase. H 0 Production by the Cyanide Cgt lyzed Oxidation of Ribulose-Pz. Spin-trapping of'OH Radical by DMPO. The Effect of Fructose-2,6-P on Ribulose-P2 Carboxylase Actiaation. The Effect of Trans-Z-hexenal on Ribulose-P Carboxylase and Oxygenase Activities. The Inhibition of Ribulose-P2 Carboxylase by Ribose-S-P. ix 96 98 102 117 122 124 126 134 136 138 142 144 147 156 159 162 Bicine CABP chl DETAPAC DMPO MnSOD NBT2+ ribulose-P2 xylulose-P LIST 9: ABBREVIATIONS N,N'-bis(2-hydroxyethyl)glycine 2'-C-carboxy-arabinitol-1,5-bisphosphate chlorophyll diethylenetriaminepentaacetic acid 5,5'-dimethyl-l-pyrroline-N-oxide the superoxide dismutase which contains manganese as a cofactor nitro blue tetrazolium D-erythro-pentulose-l,S-bisphosphate D-threo-pentulose-l,5-bisphosphate INTRODUCTION The literature concerning ribulose-P2 carboxylase/oxy- genase is overwhelming in scope but several recent reviews have summarized it effectively (Lorimer, 1981a; Lorimer and Andrews, 1981; Jensen and Bahr, 1977). Major recent devel- opments of the molecular aspects of the synthesis and assem- bly of the protein have occurred (Bogorad, 1981), and these. will be reviewed briefly. In addition, literature on the reaction mechanism of ribulose-P2 carboxylase/oxygenase will be summarized. Synthesis and Assembly of Ribulose-P2Carboxy- lase/Oxygenase. Ribulose-P2 carboxylase from higher plants is a hexadecameric protein of eight identical small subunits of 12,000 to 14,000 daltons and eight identical large sub- units of 50,000 to 55,000 daltons (Kawashima and Wildman, 1970). The enzyme is roughly spherical in shape as deter- mined by electron microscopy (Trown, 1965) and ultracentri- fugation (Paulsen and Lane, 1966). Crystallized enzyme from tobacco has been studied by x-ray diffraction analysis, and is characterized by a four-fold axis of symmetry and a plane of symmetry parallel to the four fold axis of symmetry (D4 symmetry group)(Eisenberg et al., 1978). The gene for the small subunit of ribulose-P2 carboxy- lase is located on the nuclear genome (Kawashima and Wildman, 1972). The small subunit is synthesized by free 1 cytoplasmic ribosomes as a precursor with a molecular weight of 4,000 to 5,000 daltons greater than the processed small subunit (Dobberstein et al., 1977). This precursor polypep- tide is taken up, processed and incorporated into ribulose-P2 carboxylase holoenzyme by intact chloroplasts (Chua and Schmidt, 1978). The precursor of the small sub- unit has not been detected in intact chloroplasts. Therefore processing probably occurs during or soon after transport. Sequence analysis of the 4,000 dalton peptide, (cleaved by proteolysis from the precursor) demonstrated that it is composed of 44 mostly nonpolar amino acid re- ‘sidues and occupies the N-terminus of the precursor peptide (Chua and Schmidt, 1979). This sequence may be involved in the post-translational transport mechanism, and has been de- signated as a "transit peptide" to distinguish it from the ”signal peptide" of precursors of secretory proteins. Tran- sit peptides may occur on the C or N terminus of precursors of organelle proteins which are synthesized by free cyto- plasmic ribosomes. The synthesis of the precursor is not intimately coupled to transport. In comparison, signal pep- tides occur on the N terminus of secretory proteins which are synthesized by membrane-bound ribosomes and protein syn- thesis is directly coupled to transport. The small subunit of the spinach enzyme has been sequenced by amino acid sequencing (Martin, 1979), and the small subunit of the pea enzyme has been sequenced by DNA sequencing (Bedbrook et al., 1980). The amino acid sequence of the small subunit from these sources is poorly conserved. At present it is not possible to discuss structure-function aspects of the small subunit. This subunit has no known function, except that it dramatically improves the solubility of the large subunit which is essentially insol- uble in its absence. The gene for the large subunit of ribulose-P2 carboxy- lase is maternally transmitted (Kawashima and Wildman, 1972) and the location of the gene in the chloroplast genome has been documented by physical techniques (Link and Bogorad, 1980; Bedbrook et al., 1979). The large subunit gene of maize has been cloned and sequenced, and the amino acid se- quence is highly conserved when compared to peptides iso- lated from carboxylases of most photosynthetic organisms (Bogorad, 1981). In fact, the amino acid sequence between residues 170 to 220 is essentially completely conserved in Synechococcus, a blue-green algae, Chlamydomonas, a green algae, and spinach and maize, flowering plants (Lorimer, 1983). The amino acid sequence of the large subunit from the purple sulfur bacterium, Rhodospirullum rubrum, is con- siderably less homologous when compared to the sequence of the large subunit from other photosynthetic organisms (Hartman et al., 1982). However, considerable homology does exist in several sequences which probably define the active site residues (174-180 and 326-338). The relative lack-of sequence homology between the R; rubrum and other bacterial and plant carboxylases suggests that the enzyme could have evolved independently in the two groups of organisms. This notion could provide new support for the view that ribulose-P2 oxygenase is an unavoidable consequence of the ribulose-P2 carboxylase reaction (Lorimer, 1981a). The Structure of the Catalytic Site. Ribulose-P2 car- 2+ boxylase is activated by incubation with CO2 and Mg with commensurate changes in kinetic properties (Vmax of the carboxylase and oxygenase increase, Km(C02) de- creases)(Jensen and Bahr, 1977). The protein reacts with CO2 in a slow reaction, followed by the rapid association with MgZ+(Eq. l) (Lorimer et al., 1976). slow Enz-CO fast , 2 I Enz-CO Enz -Mg2+ (Eq. 1) 2 The reaction of CO2 with a specific lysyl residue of the large subunit (lysine 201) has been verified and localized by methylation of the lysyl-carbamate, isolation of the derivitized peptide and sequence analysis (Lorimer and Miziorko, 1980). Carbamate formation occurs by the reaction of an unprotonated lysyl residue with a C02 molecule (Lorimer, 1981b). This form of the enzyme binds M92+, and several investigators have speculated that the activating metal ion may directly interact with the carbamate (Miziorko and Sealy, 1980; Pierce et al., 1980), although there is no data available on this aspect. The Mg2+ may pull the equilibrium toward activation, and this may account for the effect of Mg2+ on the pH optimum of activation (Sugiyama et al., 1968a). The distance between an activating metal ion, Mn2+ , and the substrate CO2 has been determined as 5.4 1 0.1 A by NMR spectroscopy (Miziorko and Mildvan, 1974). CABP, a tight binding inhibitor, renders the activating CO2 molecule and metal ion nonexchangeable (Miziorko and Sealy, 1980). The stoichiometry of CABP, CO2 and metal ion incorporated is 1:1:1 per protomer, and 14 C labelled CABP or CO2 may be used to measure the number of sites capable of binding these li- gands (Pierce et al., 1982). The large subunit of ribulose-P2 carboxylase/oxygenase contains the catalytic site and the activation site. Lysyl residues (175, 334) and cysteinyl residues (172, 459) have been derivitized by treatment of the enzyme from spinach with affinity ligands, and these residues may define the geography of the catalytic site (Schloss et al., 1978b). Of these four amino acid residues, only lysine 175 was derivi- tized under activating conditions (in the presence of M92+). Lysine 175 also forms a Schiff-base with pyridoxal-P (Spellman et al., 1979) or glyoxylate (Cook, Tolbert, and Hartman, unpublished). Lysine 175 exhibits enhanced nucleo- philicity toward these active site probes, and the reactiv- ity suggests that this residue could be catalytically func- tional. It is tempting to speculate that lysine 175 may be the base which abstracts a proton from C-3 of ribulose-P2 and catalyzes enolization. Hartman and colleagues have compared the susceptibility of the R; rubrum enzyme to active site probes. A lysyl re- sidue which corresponds to lysine 175 is derivitized by pyridoxal phosphate. The counterpart of lysine 334 has not been derivitized, although methionine 335 was derivitized by active site directed probes. Thus the assignment of these regions of the polypeptide as part of the catalytic site seems reasonable. The role of cySteinyl residues in the catalytic mechanism of ribulose-P2 carboxylase has been questioned since these residues (cysteine 84, 99, 172, 192, 221, 247, 427 and 459 of the spinach enzyme) are substituted in the R; rubrum enzyme (Hartman et al., 1982). Group specific reagents have been utilized by several investigators to establish the involvement of specific amino acid residues in catalysis. Arginine residues have been implicated by reaction with dicarbonyl reagents (Schloss et al., 1978a; Lawlis and McFadden, 1978); tyrosine by reaction with tetranitromethane (Robison and Tabita, 1979); cysteine by reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) (Rabin and Trown, 1964; Trown and Rabin, 1964); histidine by reac- tion with diethyl pyrocarbamate (Saluja and McFadden, 1980). Typically the only proof that a catalytic site amino acid residue was involved in the inactivation phenomenon was based on ribulose-P2 protection from inhibition. Protection by substrate is not sufficient proof that an active site amino acid is involved in these studies since substrate binding could modify the accessibility or reactivity of an amino acid which is in any part of the protein. Therefore, evidence that enzyme inactivation due to specific reaction with an active site residue has been weak in these studies. In summary, the amino acid residues which appear to have a specific role in catalysis are lysyl 201, which re- acts with CO2 during activation, and lysyl 175, which ex- . hibits enhanced nucleophilicity and reactivity with cataly- tic site directed probes under activating conditions. Other amino acids are derivitized by catalytic site probes in the absence of Mg2+ (lysyl 334, cysteinyl 173 and 458). This indicates that a comformational change occurs in the enzyme with the addition of Mg2+ and that the catalytic site may be defined by amino acid residues from three distinct portions of the polypeptide. The Reaction Mechanism of Ribulose-P2 Carboxylase/Oxy- genase. Calvin (1954) suggested that the mechanism of ribu- lose-P2 carboxylation involved enolization of ribulose-P2, nucleophilic attack on CO2 to form a six carbon carboxylated intermediate, followed by hydrolysis to two molecules of glycerate-3-P (Scheme 1, page 8). This mechanism has been essentially verified by recent reports. Other carboxylation mechanisms have been considered, such as those involving c=o HCOH HCOH CHZOPO§ “o\ ,CHzopozj _<,3H20P03 .0. (3‘0“ xc‘OH ‘Y A 9:0 HCOH HCOH CHZOPO§ zopoi co2 02 . cHZOPog cnzopog 05' '?"OH ' .0 9:0 HO-C-C\ _ H90“ . 33:0 0 cnzoeog I 4 “$0“ _ . CHZOPO§ CH20P0§ ‘O-O-CIS-OH H20 (9:0 HCOH CH OPO‘ CHZOPO§ I 2 ,,o 3 H20}; H-I—C‘o- 9H20P0§ OH ‘O’CFO . “(D-940 -O\$¢O H-C-OH H-c-OH CHzopog CHZOPO§ cHzopog Scheme 1 thiohemiketal (Rabin and Trown, 1964) or ketimine formation (Wishnick and Lane, 1969) between an amino acid residue and ribulose-P2. Ketimine formation could not be observed by reductive trapping. These mechanisms now seem unlikely be- cause the oxygen atoms at carbon two and three of ribulose-P2 are retained in the products of the carboxyla- tion reaction (Sue and Knowles, 1978; Lorimer, 1978). Enolization of ribulose-P2 by ribulose-P2 carboxy- lase/oxygenase from Rhodospirullum rubrum has been ex- tensively studied by Knowles and his associates. Activated ribulose-P2 carboxylase catalyzes the exchange of solvent protons with the carbon 3 proton of ribulose-P2 (Saver and Knowles, 1982), and tritium label is lost from carbon 3 of ribulose-P2 (Sue and Knowles, 1982a). The rates of exchange are slow relative to catalysis. These data were interpreted to indicate that enolization is a slow step in the ribulose-P2 reaction mechanism, but that enolization is not rate determining. This conclusion was partially confirmed by the primary kinetic isotope effect for 3-deuterio-ribu- lose-P2 which is 1.2 (Sue and Knowles, 1982b). This value is too small to be an isotope effect for the rate-limiting removal of the carbon 3 proton. Siegel and Lane (1973) reported that the epimeric mix- ture of 2-carboxy-arabinitol-P2 and -ribitol-P2 was inhibi? tory to-ribulose-P2 carboxylase, and suggested that a six carbon carboxylated intermediate was formed during the car- 10 boxylase reaction. ~Pierce et a1. (1980) resolved the mix- ture of epimeric carboxy-pentitol-P2 and demonstrated that 2-carboxy-arabinitol-P2 (CABP) was the species which the enzyme bound exceedingly tightly (KD= 10 pM). Since 2-car- boxy-arabinitol-Pz, not the ribitol epimer, had potent in- hibitory properties, CABP was proposed to be a transition state analogue of the carboxylation reaction. Recently, Schloss and Lorimer (1982) isolated the re- duction product of the carboxylated intermediate and con- firmed the structure of the enzyme-bound intermediate as 2-carboxy-3-keto-D-arabinitol-l,5-bisphosphate. This inter- . mediate could undergo carbon bond breakage by attack of hydroxyl ion on the carbonyl (carbon 3) with formation of two molecules of D(-)glycerate-3-P. An aspect of ribulose-P2 oxygenase which is poorly un- derstood is the mechanism of the reaction with dioxygen. Dioxygen exists in the ground state as a triplet; it has two unpaired electrons and the direction of spin for each elec- tron is the same. The products of the oxygenase reaction, glycerate-3-P and glycolate-Z-P, are singlet molecules, i.e. all electrons are paired in orbitals with opposite spin directions. The lowest singlet state of dioxygen is 22 Kcal per mol above ground state and this species is probably not involved in the oxygenase reaction. Angular momentum must be conserved in a chemical reaction, just as in all physical interactions. Therefore, over the course of the oxygenase ll reaction the angular momentum of the two spinning electrons of dioxygen must ultimately cancel each other in the pro- ducts. The electron spins may become opposite through spin inversion, a process where the direction of electron spin switches orientation, or if spatial separation occurs, the electron spins may become uncoupled and lose their original orientation. The time required for spin inversion varies between 1 to 10"9 5 depending on the environment (Hamilton, 1974), and is much slower than most chemical reactions. As a consequence of the cdnservation of the angular momentum of the spinning electrons and the slow rate of spin inversion, the direct reaction of a singlet and triplet to give singlet products is impossible. Oxygenases typically utilize either a transition metal or an organic cofacter to catalyze a reaction with 02. The d-orbitals of transition metals may overlap with the p-orbi- tal of dioxygen. The total angular momentum of the elec- trons in the metal-oxygen complex is involved, and oxygen can react by a nonradical (ionic) mechanism (Hamilton, 1969). In this case, the net angular momentum of the electrons is the same after the substrates have reacted. Organic cofactors readily react with 02 by virtue of their ability to form resonance stabilized radical intermediates. For example consider the reaction of reduced flavin with 0 (Eq. 2) (Massey et al., 1969): 2 12 Flavin-H2 + 02 ---> Flavin-H'+ O - + H+ ---> Flavin + H 02 (Eq. 2) 2 2 In this reaction, a singlet (reduced flavin) and a triplet (dioxygen) react with a one electron transfer to form two doublets (two free radicals, flavin semiquinone and superox- ide). Note that the net angular momentum has not changed, but the electron spins become uncoupled when they reside on different molecules. Subsequent reaction may occur between the two radicals, yielding oxidized flavin and hydrogen per- oxide. Therefore, it is a spin allowed process for a trip- let and singlet to react and give two doublet intermediates, which may recombine to singlet products (Hamilton, 1974). Ribulose-P2 oxygenase seems to be an anomoly among en- zymes which catalyze oxygenase reactions, since it contains no transition metal or organic cofactor (Chollet et al., 1975; McCurry, 1979; this thesis, Chapter 2). The enzyme is an internal monooxygenase, in which one atom of dioxygen is incorporated into product (the carboxy of glycolate-Z-P) and one atom into H20 (Lorimer et al., 1973). An oxygen atom from dioxygen is not incorporated into the carboxy of gly- cerate-3-P; thus formation of a dioxetane ring with carbons two and three during oxygenation seems unlikely. The enzyme has been reported to contain copper and iron, although these metals may be removed to a fraction of the enzyme active site concentration without commensurate loss in catalytic activity (McCurry, 1979). The enzyme also contains a trace 13 of flavin (Chapter 2), although this was present at far less than 1:1 stoichiometry. In summary, there is very good evidence that ribulose-P2 lacks transition metals or organic cofactors at stoichiometric levels. Two mechanisms for ribulose-P2 oxygenase have been pro- posed (Lorimer, 1981a; Lorimer and Andrews, 1981). Scheme 2 (page 57) shows a possible mechanism for ribulose-P2 oxygen- ase. The enediol(ate) may tautomerize to the carbanion and this species may reduce dioxygen by one electron, indicated by the single headed arrow. The intermediates of this reac- tion would be a carbon based radical and superoxide radical, and these two radicals may recombine to form 2-peroxy-3- keto-pentitol-Pz. This putative intermediate of the oxy- genase reaction may decompose by addition and elimination of hydroxyl ion. This mechanism involves the reaction of a triplet and a singlet to form two doublets, followed by a reaction to form two singlets, essentially as discussed with the reduction of dioxygen by reduced flavin. Thus ribulose-P2 is proposed to react in a manner analogous to flavin. Further discussion of the mechanism of ribulose-P2 oxygenase is deferred to Chapter 2. The Role of Ribulose-P2 Carboxylase/Oxygenase in Photo- synthetic Carbon Metabolism. Ribulose-P2 carboxylase/oxy- genase mediates the flux of carbon between two cycles, the reductive photosynthetic carbon cycle and the oxidative photosynthetic carbon cycle. If the kinetic parameters for l4 ribulose-P2 carboxylase and oxygenase could be modified, perhaps photosynthesis and crop productivity could be improved. Therefore many investigators have attempted to modify the ratio of carboxylase to oxygenase activities. Hydroxylamine has been reported to selectively inhibit ribu- lose-P2 oxygenase (Bhagwat et al., 1978a; Okabe et al., 1979), although this could not be repeated in other labora- tories (Brown et al., 1980; Mulligan, unpublished). Super- 2+ have been reported to inhibit oxide dismutase and NBT ribulose-P2 oxygenase (Bhagwat and Sane, 1978b), and this too could not be repeated in our laboratory (McCurry, 1979; this thesis, Chapter 2). Aliphatic aldehydes have been re- ported to selectively inhibit ribulose-P2 oxygenase (Martin and Tabita, 1982). but selective inhibition was not observed with either glyoxylate or trans-Z-hexenal in our laboratory (this thesis, Chapters 3 and 7). The reasons for the fre- quent discrepancies in results from different laboratories are unclear, but may relate to the procedures used to assay the enzyme. For example, an artifact could be generated by preincubation of ribulose-P2 with hydroxylamine to form the corresponding hydroxamate. Different periods of preincuba- tion for the carboxylase and oxygenase assays might result in different concentrations of substrate, and a differential effect might be suggested due to this artifact. Similarly, time-dependent inactivation by aldehydes might cause a similar artifact if aldehyde is preincubated with enzyme for 15 different periods before assaying for carboxylase or oxygen- ase activities. The ratio of ribulose-P2 carboxylase to oxygenase is dependent on the relative concentrations of CO2 and 02, since these gases are linearly competitive inhibitors with respect to each other (Laing et al., 1974). C4 plants and some algae utilize this phenomenon by concentrating CO2 at the site of carboxylation and consequently suppress the oxy- genase activity and photorespiration. The activation ener- gies of the carboxylase and oxygenase activities differ, and therefore the ratio of carboxylase to oxygenase varies with temperature (Badger and Collatz, 1977). The metal ion in- cluded in activation and assay modifies the kinetic para- meters of the enzyme (Christeller, 1981). Enzyme activated with Mn2+ exhibits a greatly decreased Km(02) and modified ratio of carboxylase to oxygenase. No_compound which has been tested has selectively inhibited the oxygenase, and the ratio of carboxylase to oxygenase activity from any given source is constant within the constraints indicated above. An important point is that the ratio of carboxylation to oxygenation does vary when the carboxylase from various sources is surveyed (Jordan and Ogren, 1981). These authors claim that the kinetic properties of the enzyme are mutable and therefore may be manipulated genetically. Although the ratio of carboxylase to oxygenase varied widely from pro- karyotes to higher plants, little difference existed in --15 higher plant carboxylases. The contention that the kinetic properties of the enzyme vary and are therefore mutable seems sound. Whether genetic manipulation may further im- prove the specificity of the enzyme for carboxylation must await proof. The role that ribulose-P2 carboxylase/oxygenase plays in plant metabolism has been firmly established. It is re- sponsible for photosynthetic carbon fixation and for initiating photorespiration. The enzyme shows changes (1-4 fold) in activity with dark to light transitions (Bahr and Jensen, 1978), but these changes are small relative to the 20 to 40 fold activation of ribulose-S-P kinase, fructose- bisphosphatase and sedoheptulosebisphosphatase (Laing et al., 1981). Therefore, the physiological significance of CO2 and Mg2+ activation has not been clearly demonstrated and remains controversial. Ribulose-P2 oxygenase catalyzes a reaction which initiates glycolate synthesis. The source of glycolate has been very controversial. Oxidation of the dihydroxyethyl derivative of thiamine pyrophosphate (an intermediate of the transketolase reaction) by H202 has been fostered by some as the source of glycolate (Shain and Gibbs, 1971). This reac- tion is exceedingly slow and is unable to account for physiological rates of glycolate formation (Christen and Gasser, 1980). In addition, a mutant of Arabidopsis which lacks glycolate-Z-P phosphatase will grow under low 02 ten- T7 sions, but the lesion is lethal under photorespiratory conditions (Somerville and Ogren, 1979). This mutant accumulates glycolate-Z-P, even when glycolate oxidase is inhibited by a suicide inactivator. At present, it is generally agreed that ribulose-P2 oxygenase initiates glycolate synthesis and is principally responsible for photorespiration. Ribulose-P2 oxygenase is not solely responsible for light-dependent O2 Consumption. Photosystem I carries out the reduction of O2 and this phenomenon is termed psuedo- cyclic electron transport or the Mehler reaction (Marsho et al., 1979). Under ambient CO2 and O2 tensions, it has been - estimated that 65% of the dioxygen consumption is due to photorespiratory uptake, presumably via ribulose-P2 oxygenase and that the remaining 02 consumption is due to 02 reduction (Behrens et al., 1982). The outstanding question in the field of photorespira- tory carbon metabolism is: Why does this phenomenon exist? The process was proposed to be wasteful, since several oxidative reactions are involved which are not coupled to pyridine nucleotide reduction or phosphorylation (Andrews and Lorimer, 1978). A related viewpoint proposed that ribu- lose-P2 oxygenase is an unavoidable consequence of the reac- tion mechanism of ribulose-P2 carboxylase (Lorimer and Andrews, 1973b). This may be true whether photorespiration has a useful role or is a wasteful process. A third view- 18 point Suggests that photorespiration is a means for the orderly dissipation of excess reducing equivalents (Powles et al., 1979; Heber and Krause, 1980). Leaves exposed to 2's-l) in the absence high irradiant flux (2000 pEinsteins'm- of CO2 and reduced 02 tension showed decreased rates of photosynthesis (Powles et al., 1979). Leaves exposed to the same light intensity with CO2 at the compensation point or atmospheric O2 tension suffered essentially no photoinhibi- tion. Thus a plant appears to suffer irreversible inhibi- tion of the photosynthetic apparatus when exposed to light in the absence of electron acceptors. Photosystem II is sensitive to photoinhibition (Powles et al., 1979) and addi- tion of CO2 provides a sink to accept reducing equivalents and phosphorylation energy from the light reactions. Similarly O2 stimulates the oxidative photosynthetic carbon cycle which effectively consumes reducing equivalents and phosphorylation energy. Thus, either C02 or 02 may provide a sink for electrons from the light reactions and protect the plant from photoinhibition. Although this rationale for the existence of photorespiration is not yet well document- ed, the model predicts an important physiological role for photorespiration, which is amenable to experimentation and is substantiated by the limited literature on this topic. CHAPTER 1 THE LABILITY OF AN INTERMEDIATE OF THE RIBULOSE-Pz CARBOXYLASE REACTION The mechanism of ribulose-P2 carboxylase was probed by libera- tion of an enzyme-bound intermediate through interruption of the catalytic cycle by acid denaturation. This technique was used by Iyengar and Rose (1981a,b) to study the ene-diol intermediate of the reaction catalyzed by triose-P isomerase. In their study, acid treat- ment of the enzyme-substrate complex liberated an intermediate with a labile phosphate ester. Schloss and Lorimer (1982) utilized this technique for the reductive trapping of the carboxylated intermediate of the ribulose-P2 carboxylase reaction. In this study, acid denaturation of ribulose-P2 carboxylase/oxygenase also liberated an intermediate with a labile phosphate ester. The intermediate is probably the carboxylated intermediate rather than the ene-diol(ate) intermediate. A manusCript with results fron this chapter has been accepted for publication in Archives of Biochemistry and Biophysics. 19 20 Materials and Methods Ribulose-Pz was prepared from ribose-S-P and ATP as described by Horecker et al. (1956) and purified by chromatography on Dowex 1-Cl (X8) with a linear 0-0.4 M LiCl gradient prepared in 5 mM HCl. The fractions c0ntaining ribulose-P2 were determined by analysis for alkaline labile P1 or by enzyme assay with ribulose-P2 carboxylase. These fractions were pooled and the ribulose-P2 was precipitated as the barium salt in the presence of 50% ethanol. The precipitate was washed by reSUSpension in 95% ethanol and lyophilized. For the synthe- sis of [1-32PJribulose-P2, [Y-32PJATP was prepared from 32P1 and unlabeled ATP by the exchange reaction catalyzed by glycerate-B-P kinase and glyceraldehyde-3-P dehydrogenase (Glynn and Chappel, 1964). Inorganic phosphate contamination of the ribulose-P2 preparations was 3-5 % of the ribulose-P2 concentration. The barium salt of ribulose-P2 was dissolved by treatment with Dowex SO-HT. Ribulose-Pz solutions were converted to the Na+ salt by treatment with Dowex 50-Na+, by titration with 0.1N NaOH with rapid stirring, or converted to the triethanolamine salt by titration with 1 M triethanolamine. The pH of ribulose-P2 solutions was maintained at less than 6.5 for stability, and ribulose-P2 was added to buffered reaction mixtures (pH 8.2) immediately before use. The dimethyl ketal of DHAP was obtained from Sigma and converted to the keto form according to the instructions provided. Xylulose-Pz was prepared as previously described (McCurry and Tolbert, 1977). Other chemicals were of reagent quality and used without further purification. Ribulose-Pz carboxylase/oxygenase was purified from Spinacia oleracea L, leaves, which were obtained from a local market. The 21 purification procedure involved ammonium sulfate fractionation, molecular sieve chromatography and ion exchange chromatography as described elsewhere (McCurhy et al., 1982). The enzyme was precipi- tated with 50% saturated ammonium sulfate and frozen into pellets by dropwise addition to liquid N2 and stored in a -80°C freezer. Aldolase (rabbit muscle) and other enzymes were obtained from Sigma and were dialyzed exhaustively before use. Protein concentration was determined by the absorbance at 280 nm (absorbance x 0.61 = concentration in mg/ml) (McCurry et al., 1982). Enzyme active site concentrations were calculated by dividing the protein concentration (mg/ml) by the molecular weight of a carboxylase protomer (68,750 daltons). Ribulose-Pz carboxylase/oxygenase assays were performed as described elsewhere (Pierce et al., 1982). The enzyme was activated with 10 mM NaHC03, 20 mM MgCl2, 1 mM dithiothreitol, 0.2 mM EDTA and 100 mM Bicine-Na+ (pH 8.2) forflatflleast 30 min at 30°C.” The ‘ I”. a‘“..,_....~-m ”mu—nu“ ’ carboxylase activity was assayed by the addition of enzyme (4-100 ug) to a 0.25 or 0.50 ml assay mixture containing 0.5 mM ribulose-P2, 10 mM NaH14CO3 (30.14 Ci/mol), 20 an M9012, 1 mM dithiothreitol, 0.2 mM EDTA, and 100 mM Bicine-Na+ (pH 8.2). Assays were terminated by the addition of 0.1 or 0.2 ml of 2 N HCl, reduced to dryness at 95°C, and analyzed for 14C by liquid scintillation counting. Oxygenase assays were initiated by the addition of 160 ug of activated enzyme to a 1 ml assay mixture containing 0.5 mM ribulose-P2, 100 nM Bicine—Na+ (pH 8.2), 20 mM M9012 and 0.2 mM EDTA. The initial rate of 02 consumption was followed polarigraphically with a Rank Brothers oxygen electrode. 22 Experiments which interrupted the enzyme catalyzed reaction at approximately one turnover were performed using one of two procedures. In one technique, activated ribulose-P2 carboxylase/oxygenase (0.25 ml) was added to a rapidly stirred solution of ribulose-P2 (0.25 ml) in 100 mM Bicine-Na+ (pH 8.2). The ribulose-P2 solution contained twice the number of moles of ribulose-P2 as the number of moles of enzyme active sites which would be added. Catalysis was terminated after approximately 0.5 s by the addition of 0.1 ml of 1.2 N trichloroacetic acid. In control reactions, the acid was added to the enzyme before mixing with the ribulose-P2 solution. Denatured protein was removed by centrifugation and an aliquot was withdrawn for Pi determination. Phosphate was determined spectrophotometrically by an ultrasensitive procedure which was based on the ability of the phosphomolybdate to complex with malachite green (Tashima and Yoshimura, 1975). The colorimetric reagent was prepared by mixing one volume of 3% (w/v) ammonium molybdate with one volume of 0.06% (w/v) of malachite green-HCl in 6 N HCl. Occasionally it was necessary to filter this solution before use. The sample was diluted to 1 ml, and 0.5 ml of the colorimetric reagent was added. The solutions were mixed and the absorbance at 650 nm was determined after 15 min at room temperature. The absorbance was linear up to 10 nmol of Pi with a molar extinction coefficient of 9 x 104. Since the procedure described above was subject to considerable variability of the reaction time, a simple, rapid mix and quench device was constructed based on the principles outlined by Froehlich et al. (1976). An aluminum block with two grooves (0.5 cm deep) held two 1 ml disposable tuberculin syringes. A plexiglass top with 4 wingnuts 23 clamped the syringes in place. A hand operated drive plate provided .simultaneous and constant flow to the two syringes. Each 1 ml syringe was fitted with an 18 gauge needle which was connected to 1/32" inside diameter (1.0.) silicone tubing (Dow Corning). The solution from each syringe was divided into two with the use of a tee connector and each side was joined to a second tee connector with 1/32" tubing. Each second connector mixed one half of the flow of one syringe with one half of the flow of the other syringe. The solutions then flowed into 5/32" 1.0. tubing to facilitate turbulent mixing. The two solutions, which were mixed at the second tee connector, were mixed together and combined in a final tee connector, also followed by 5/32" tubing. The mixed solution then passed into 1/32" tubing which could be varied in length for different reaction times. The reaction was terminated as the mixed solutions passed into a vial containing 0.28 ml or 0.5 ml of 2 N trichloroacetic acid. The acid solution was rapidly stirred with a magnetic stirbar. The apparent reaction time was calibrated by the base catalyzed hydrolysis of 2,4-dinitrophenyl acetate as described by Froehlich et al. (1976). An apparent reaction duration of 150 ms was used in these experiments. In a rapid mix and quench experiment, [1-32PJribulose-Pz was loaded in one syringe in 0.70 ml of solution containing 70 pH ribulose-P2, 100 mM Bicine—Na+ (pH 8.2), 20 mM MgCl2, and 10 mM NaHCO3. Ribulose-Pz carboxylase/oxygenase in activation solution was loaded in the other syringe in 0.70 ml of solution containing 35 uM active sites.” In control experiments, enzyme was omitted from the activation solution. The reaction was terminated by passage of the reaction mixture into trichloroacetic acid as describedabove. An 24 aliquot was taken from the terminated reaction mixture and analyzed for 32P; by extraction of the phosphomolybdate complex into isobutanol as described by Berenblum and Chain (1938). Alkaline labile phosphate, representing unreacted ribulose-P2, was determined by addition of NaOH to a final concentration of 0.5 N and incubation at 37°C for 15 min. This sample was then acidified and extracted for P1. Results Phosphate Lability of an Enzyme Liberated Intermediate. The alkaline lability of phosphate esters which are 8 to a carbonyl carbon is well established. Ribulose-Pz is known to eliminate Pi under alkaline conditions and rearrange to a compound which is probably 1-deoxy-2,3-diketo,pentitol-5-P (Paech et al., 1978). A similar reaction is catalyzed by triose-P isomerase. Iyengar and Rose (1981a,b) reported that triose-P isomerase catalyzes the formation of the ene-diol form of dihydroxyacetone-P, an enzyme-bound intermediate, which may subsequently rearrange to methylglyoxal by Pi elimination. Iyengar and Rose extended this observation by liberation of the ene-diol from the active site through acid treatment of the enzyme, and approximately 5% of the enzyme-bound phosphate ester was labile. Since the reaction mechanism of ribulose-P2 carboxylase/oxy- genase is pr0posed to proceed via an ene-diol(ate) intermediate, the ability of this enzyme to catalyze the formation of an intermediate with a labile phosphate ester was examined. Activated ribulose-P2 carboxylase/oxygenase (9 to 105 nmol of active sites) was added to a. 25 rapidly stirred solution of ribulose-P2. The reaction was quenched with 0.1 ml of 1.2 N trichloroacetic acid after approximately 0.5 5 under these conditions. This protocol should quench the enzyme cata- lyzed reaction during steady state catalysis since the turnover time is approximately 0.5 5 under these conditions. More P1 was present in the reaction mixture when enzyme reacted with substrate than in the control, in which acid was added to the enzyme before the substrate (Fig. 1). Linear regression of the P1 released as a function of the amount of enzyme present indicates that the P1 released was equivalent to about 5% of the enzyme active sites. With the hand quench technique, ribulose-P2 carboxylase/oxygenase activated with 1 mM MnClz also liberated Pi- When xylulose-1,5-P2 was mixed with ribulose-P2 carboxylase, no P1 was liberated. Xylulose-1,S-P2 is a competitive inhibitor with respect to ribulose-P2 in the ribulose-P2 carboxylase/oxygenase reaction (McCurry and Tolbert, 1977). when 70 an [1-32PJribulose-P2 was mixed with 35 an active sites of ribulose-P2 carboxylase and quenched with acid after approximately 150 ms, additional 32Pi was extractable which was equivalent to 4.1 1 0.7% of the enzyme active site concentration (mean 2 S.E., 6 determinations). Analysis of the unreacted ribulose-P2 by extraction of P1 after alkaline treatment indicated that each active site turned over an average of 0.13 times. In an experiment in which the enzyme catalysis was allowed to go to completion, no P1 was liberated in the normal course of catalysis. Thus P; liberation is totally dependent on the interruption of the catalytic cycle, and was not a phenomenon associated with normal catalysis. Figure 1. 26 Phosphate Liberation as a Function of the Amount of Ribulose-Pz Carboxylase Active Sites. Activated enzyme (9 to 105 nmol of active sites in 0.25 ml) was added to a rapidly stirred solution (0.25 ml) of 0.1 M Bicine-Na+ (pH 8.2) containing twice the number of mol of ribulose-P2 as active sites added. Trichloroacetic acid (0.2 ml of 1.2 N) was added after about 0.5 s. Phosphate was determined by the malachite green procedure as described in materials and methods. The line calculated by linear regression has a slope of 0.048 1 0.009 (mean t S.E.). nMol P. Released 27 T l T r r 20 40 60 80 100 120 nMol Active Sites Figure l. The intermediate with the labile phosphate ester is probably either the ene-diol(ate) or the carboxylated intermediate. The ene-diol intermediate liberated from triose-P isomerase by acid denaturation is rapidly labile, with a half life for phosphate elimination between 12 and 17 ms (Iyengar and Rose, 1981b). Therefore the stability of the liberated intermediate from the carboxylase reaction may provide some information regarding its identity. Schloss and Lorimer (1982) stabilized the carboxylated intermediate by reduction with NaBH4. A similar experiment was designed to stabilize the labile intermediate after a brief period in acid. However, treatment of the acid quenched reaction with buffered NaBH4 interferred with subsequent extraction of 32Pi; destruction of the NaBH4 with glucose or acid did not alleviate the interference. Another approach to examine the stability of the liberated intermediate was to enzymatically convert it to the final products of the carboxylation reaction. Five seconds after acid treatment, an additional 0.5 ml of activation buffer with or without 4.12 mg of purified spinach carboxylase was added to the acid quenched solution (Table 1). The addition of a second aliquot of enzyme effectively prevented the liberation of Pi- These results indicate that the labile intermediate was stable for 5 s in acid. Furthermore the liberated intermediate was apparently a substrate for the enzyme catalyzed reaction, since addition of buffer alone did not prevent the release of Pi- The relatively stable nature of the intermediate suggests that Pi release was not from the ene-diol(ate) which should have eliminated P1 or tautomerized to the stable keto form within 5 s. 29 Table 1. The Stability of the Phosphate Labile Intermediatea. Pi released, expressed as % Ribulose-PZ Post Quench Treatment % of the active sites Remaining None 5.0 93.5 Activation Buffer 4.4 90.7 (No Enzyme) Activated Enzyme 0 0 aRibulose-Pz and ribulose-P2 carboxylase were mixed for approxi- mately 150 ms and quenched in trichloroacetic acid (final concentration of 0.08 N). The post quench solution was added after 5 s, and contained 250 mM Bicine-Na+ (pH 8.5), 1 mM dithiothrieitol, 20 mM M9012, 10 mM NaHCO3 with or without 8.23 mg mi-1 of purified spinach carboxylase. Pi and unreacted ribulose-P2 were determined after 1h at 22°C. 30 To establish the lability of the carboxylated intermediate, the reductive stabilization technique of Schloss and Lorimer (1982) was used. In our experiment, [1-32PJribulose-P2 and 14C02 were mixed with activated enzyme for approximately 150 ms and quenched with HCl. After 5 s or 1 h at 22 C, the acid quenched solution was reduced with NaBH4 and chromatographed on Dowex l-Cl. The elution profiles obtained after NaBH4 reduction after 5 s or 1 h are shown in Figure 2A and 28 respectively. Peak 2 co-chromatographed with glycerate-3-P prepared enzymatically with ribulose-P2 and carboxylase. _The ratio of 32le4c in peaks 2 and 4 was the same, and the radiospecific activities indicated that each peak contained compounds which incorpor- ated C from C02 and P from ribulose-P2 with a ratio of 1:1. Peak 4 chromatographed similarly to the lactone of carboxyarabinitol-P2. Thus peak 4 was due to the redUction product of the carboxylated intermediate. The 14C observed in peak 4 of Figure 2A represented 4.2 nmol of carboxylated intermediate which was trapped from 112 nmol of active sites. Peak 3 was mostly pentitol-Pz, the reduction product of unreacted ribulose-P2. This peak also contained a small amount of 14C which may be due to the incomplete lactonization of carboxyarabinitol-P2 by the procedure described by Schloss and Lorimer (I982). Considerable correction for the spill of 32P counts into the 14C channell was required for the large number of counts from 32P in peak 3. Therefore, the 14C counts observed in the peak 3 fractions were subject to considerable error. A comparison of Figures 2A and 28 indicates that the carboxylated intermediate was trapped only in the sample in which reduction was initiated 55 after denaturing the enzyme with acid. After 1 h, only a Figure 2. 31 Resolution of the Carboxylated Intermediate from the Ribulose-Pz Carboxylase Reaction after Acid Denaturation and Reduction. Activated ribulose-P2 carboxylase (112 nmol active sites) was mixed with a ribulose-P2 solution for approximately 150 ms and delivered to a quench solution of 2.1 ml of 0.2 N HCl. ~Five ml syringes were used in this experiment to accomodate 1.75 ml of enzyme and ribulose-P2 solutions. The reaction mixture contained 70 mM Bicine-Na+ (pH 8.2), 10 mM NaH14co3 (0.7 uCi-umoi-l), 20 mM MgCl2, 1 mM dithiothreitol, 2.65 mg-ml'1 spinach ribulose-P2 carboxylase and 40 uM [1-32P]ribulose-P2 (0.5 uCi-umol'l). In Figure 2A, 10 ml of a solution containing 0.1 M NaBH4 and 0.1 M NaHCO3 was added 5 s after acid denaturation. In Figure 28, the NaBH4 solution was added after 1 h. Subsequent sample preparation and chromatography is described by Schloss and Lorimer (1982). Briefly this involved removal of borate, lactonization of carboxyarabinitol-P2 and chromatography on Dowex l-Cl (X8) with a linear gradient of 0-0.8 M LiCl prepared in 5 mM HCl. Aliquots (0.5 ml) were counted for 32P and 14C. iiiii Ego .a -iiiiEoo .n. 32 NM. N coon—Z DOA—um Down DOM: oowN Doom: 02.90 comm n ODD¢ ODON o . . m 10 6 4 i0 5 (0 _ 4 3- n H H iiiiiiiiiiiiiiiiiiiii A 2...: . o . _ . . _ . 0 Doom Q . OOON can: 606' Doom OOON 69.: o E 0 0 4 EQO .0 ¢P , .3 Fraction Number Figure 2. 33 Figure 3. Inhibition of Ribulose-PZ Carboxylase by the NaBH4 Reduction Product of the Carboxylated Intermediate. A) Aliquots (0.1 ml) of fractions of peak 4 from Figure 2 were incubated at pH 9.0 for 12 h at 22°C to hydrolyze the lact- ones and then added to 0.05 nmol of activated ribulose-P2 carboxylase protomers. After 2 h incubation, the enzyme was assayed for carboxylase activity in a volume of 1.0 ml by the addition of 1 mM ribulose-P2. The specific activity of the enzyme was 1.8 umol-min"1-mg'1 protein. The solid line is for the gradient fractions in Figure 2A (reduction after 5 s) and the dashed line is for fractions of the gradient shown in Figure 28 (reduction after 1 h). B) The 14C in each fraction from Dowex chromatography is shown. The solid line is from Figure 2A (reduction with NaBH4 after 5 s) and the dashed line is from Figure 28 (reduction after I h). 34 A _ _ . q 00. Om on Ox. 8 axe 53:8. omsxxoncoo 952$. Frocrion Number Figure 3. 35 trace of the carboxylated intermediate was trapped. In Figure 28, the loss of 32P counts due to the carboxylated intermediate (peak 4) after 1 h incubation was accompanied by an increase in the 32P counts due to P1 (peak 1). Further evidence that the carboxylated intermediate could be trapped by reduction after 5 s in acid, but not after 1 h, is presented in Figure 3. Aliquots of fractions from peak 4 (Fig. 2A and 28) were incubated with purified spinach ribulose-P2 carboxylase under conditions in which carboxyarabinitol forms an essentially irreversible complex with the enzyme (Pierce et al., 1980). Substantial enzyme inhibition was observed only by aliquots from the gradient presented in Figure 2A, in which reduction was initiated after 5 s. Essentially no inhibition was observed by aliquots from the gradient presented in Figure 28 in which reduction was initiated after ' 1 h. Absence of Paracatalytic Inactivation of Ribulose-P; Carboxylase/Oxygenase. Aldolase catalyzes the formation of a carbanion intermediate of dihydroxyacetone-P which is oxidized by hexacyano- ferrate (III) and results in the 2 electron oxidation of the carbohy- drate and commensurate paracatalytic inactivation of the enzyme (Christen, 1977; Christen et al., 1976). Similar studies were used to study the mechanism of ribulose-P2 carboxylase/oxygenase. The ene-diol or carbanion intermediate should have the electronic propen- sity to reduce strong oxidants such as hexacyanoferrate (III) and a preliminary report suggests that the enzyme from Pseudomonas facilis does show paracatalytic inactivation (Hsu and Kuehn, 1978). Hexacyano- ferrate (III) and DCPIP were tested for reduction by activated ribulose-P2 carboxylase/oxygenase under conditions that favored 36 carboxylase (10 mM NaHC03) or oxygenase (0.04 mM NaHC03) activities. No dye reduction could be observed spectr0photometrically and no inhibition of dioxygen consumption was observed upon the addition of these oxidants to an oxygenase assay. A possible explanation is that the active intermediate might be compartmentalized away from the oxidant. The experiment was repeated under various conditions that might have partially denatured the enzyme: 50°C; 0.2 M NaCNS; 0.2 M guanidine HCl; 0.8 M urea; and 1% Triton-X-100. Oxidant reduction could not be observed under any of these conditions. Hexacyanoferrate (III) dependent paracatalytic inactivation of ribulose-P2 carboxylase activity was also tested and no time dependent inactivation was observed with the enzyme from spinach leaves. Paracatalytic Inhibition of Fructose-P7 Aldolase. Since ~aldolase catalyzes the formation of a reactive carbanion and a reactive enediol(ate) or carbanion are proposed to be involved in the reaction mechanisms of ribulose-P2 carboxylase/oxygenase, aldolase was tested to determine if the carbanion intermediate had any propensity to undergo carboxylation or oxygenation. No carboxylase activity could be observed when aldolase (1.8 units) and dihydroxyacetone-P (1.7 mM) were incubated with NaH14C03 (1.5 uCi, 56 mCi/mmol), either carrier free or with 10 mM NaHC03. No oxygenase activity could be observed by 02 consumption under these conditions. Hexacyanoferrate (III) was readily reduced under these conditions. In the course of these experi- ments, it was observed that upon the addition of hexacyanoferrate (III) (0.5 (M) to aldolase (2 units) and dihydroxyacetone-P (1.7 mM), 02 consumption ensued at approximately 3% of the rate of hexacyanoferrate 37 reduction. Dioxygen consumption ceased when hexacyanoferrate (III) was consumed. Hexacyanoferrate (II) was not responsible for 02 consump- tion since addition of this compound did not result in 02 consump- tion. The 02 consuming process was not associated with the paracatalytic inactivation phenomenon, since the rate of inactivation of aldolase by hexacyanoferrate (III) in the presence of fructose-P2 was essentially the same under atmospheres of 100% dinitrogen or dioxygen. These results are consistent with previous results of Christen et al. (1976) where they observed that anaerobiosis did not effect the rate of aldolase inactivation. Discussion Acid denaturation-of the ribulose-P2 carboxylase reaction liberated the carboxylated intermediate which may be stabilized by reduction and isolated by anion exchange chromatography. The reduction products of the carboxylated intermediate were isolated by this procedure and identified by Schloss and Lorimer (1982) as a mixture of 2 and 4 carboxyarabinitol-P2. The compounds which were isolated after reduction are characterized to have the chromatographic properties of CABP by anion exchange chromatography (Fig. 2) and possess the potent inhibitory properties of CABP in an enzyme assay (Fig. 3), as was observed by Schloss and Lorimer (1982). In addition, the C and P incorporated into the carboxylated intermediate from C02 and ribulose-P2 was in a ratio of 1:1, which is consistent with the molecular formula of the carboxylated intermediate. The stoichiometry of the carboxylated intermediate which was trapped per enzyme active 38 site was 0.04, in reasonable agreement with the value of 0.07 obtained by Schloss and Lorimer. These data confinn the results and interpre- tation of Schloss and Lorimer that the carboxylated intermediate has the threo stereochemistry (analogous to the arabinitol configuration). The carboxylated intermediate has consequently been identified as 2-carboxy-3-keto-D-threo-pentitol-1,5-bisph05phate. Since the products of the carboxylation reaction are 2 molecules of D(-)glycerate-3-P, hydrolysis of the carboxylated intermediate must occur with inversion of the stereochemistry at carbon 2 as discussed by Pierce et al. (1980). Acid denaturation of the ribulose-P2 carboxylase reaction liberates an intermediate which has a labile phosphate ester. Since a-elimination of Pi readily occurs from ene-diol(ate)s, this form of ribulose-P2 was initially assumed responsible for the phosphate release phenomenon. However several observations suggest that the source of the inorganic phosphate is from the carboxylated intermediate. First the ene-diol of dihydroxyacetone-P was rapidly labile, with a half life of 12 to 17 ms (Iyengar and Rose, 1981a). In contrast, the labile species from the ribulose-P2 carboxylase reaction was stable for 5 s in acid (Table 1), which was considerably more stable than might be expected for an ene-diol. Secondly, the carboxylated intermediate was stable for 5 sec, but not for 1 h under acidic conditions (Fig. 2A and 28). Therefore both the carboxylated intermediate and the species with a labile ph05phate ester were stable for 5 s in acid, but not for 1 h. The instability of the carboxylated intermediate was unexpected. The mechanism of the degradation of the carboxylated intermediate is not known, but could involve 39 decarboxylation to the ene-diol with subsequent B-elimination of the carbon 1 ph05phate ester. Finally the stoichiometries of the carboxylated intermediate trapped and phosphate released to enzyme active sites were both about 0.04. Therefore the intermediate responsible for the release of phosphate is probably the carboxylated intermediate, although this has not been unequivocally demonstrated by these results. The carboxylated intermediate appears to be a substrate for ribulose-P2 carboxylase/oxygenase, since treatment of the acid quenched reaction with fresh enzyme converted the labile intermediate to a stable form. Similarly, triose-P isomerase was capable of trapping the ene-diol of dihydroxyacetone-P and the enzyme acted catalytically to convert the ene-diol to a stable form (Iyengar and Rose, 1981b). The ability of ribulose-P2 carboxylase to bind the intermediate is not unexpected since the enzyme readily binds carboxyarabinitol-P2 and other carboxylated pentitol-Pz. The carboxylated intermediate accounts for approximately 4% of the bound substrate, and very little of the ene-diol(ate) form of the substrate appears to accumulate. Perhaps this is an indication that the carboxylated intermediate exists at a lower energy level than the ene-diol(ate) in the free energy profile of the carboxylase reaction. Since only a small fraction of bound substrate exists as the ene-diol(ate) or carboxylated intermediate, the major fonn of enzyme-bound substrate probably exists as ribulose-P2 and glycerate-3-P. ‘ No P1 was liberated in the normal course of catalysis by ribulose-P2 carboxylase/oxygenase. Triose-P isomerase exhibits an 40 apparent phosphatase activity, although Pi elimination occurs at a low rate (0.1 5'1) compared to catalysis (4 x 103 5‘1) (Iyengar and Rose, 1981b). This frequency is easily observed with triose-P isomerase since the reaction is readily reversible. The carboxylase reaction is irreversible, and if Pi liberation occurs at a frequency similar with that observed with triose-P isomerase, it would have been below the level of detection. The observation that 02 consumption occurs at about 3% of the rate of hexacyanoferrate (III) reduction by the aldolase-dihydroxy- acetone-P carbanion is consistent with the oxidation occurring with a free radical intermediate (Christen et al., 1976). Since hexacyanoferrate (III) is a univalent oxidant, it is probable that the oxidation involves two single electron oxidations of the carbanion. Dioxygen is known to react readily with carbon based radicals. It is possible that the mechanism of dioxygen consumption in this system is the single electron oxidation of the carbanion to the radical, followed by reaction with dioxygen to fonn the peroxy radical. The reaction with oxygen is apparently not involved in the paracatalytic inactivation phenomenon, since rates of inactivation were not effected by equilibration with atmospheres of 100% dinitrogen 0r dioxygen. The oxygenase activity may be an unavoidable consequence of the carboxylase mechanism (Lorimer and Andrews, 1973b). Perhaps the strongest evidence for this is the ubiquitous coincidence of the two activities, even in the ribulose-P2 carboxylase enzyme from obligate anaerobes. Carbanions have been observed in certain cases to undergo reactions with both carbon dioxide and dioxygen (Lorimer and Andrews, 1973b). The experiments with dihydroxyacetone-P and aldolase indicated 41 that the carbanion intermediate of dihydroxyacetone-P does not readily undergo a reaction with carbon dioxide or dioxygen, even though the carbanion is easily accessible to solutes such as hexacyanoferrate. Therefore, it seems that something more than mere carbanion formation is required for reaction with oxygen or carbon dioxide. Although the role of metal ions in catalysis is not known it has been suggested that the metal ion in the carboxylase reaction may act to stabilize the carboxyl group of the six carbon intermediate (Pierce et al., 1980). Divalent metal ions are also known to stabilize 02- (Sawyer and Gibian, 1979) and this may relate to the conditions required for oxygenase and carboxylase activities and explain the modified kinetics in the presence of various divalent metal cations (Nildner and Henkel, 1978; Christelier, 1981). CHAPTER 2 INVESTIGATIONS ON THE MECHANISM OF RIBULOSE-P OXYGENASE. 2 Ribulose-P2 carboxylase/oxygenase is an unusual oxygenase since the enzyme is an internal monooxygenase, yet does not require a transition metal or organic cofactor for activity. An early report indicated that ribulose-P2 car- boxylase contained one mol of copper per mol of holoenzyme (Wishnick et al., 1969), but results from other laboratories indicate only trace quantities of copper associated with the enzyme (Chollet et al., 1975; Johal et al., 1980; Lorimer and Andrews, 1973a). McCurry (1979) reported that 2 mol iron per mol of holoenzyme were present in several prepara- tions of ribulose-P2 carboxylase. Dialysis against 100 mM DTT removed 65% of the iron from the protein with no loss in carboxylase or oxygenase activity. Other investigators have searched for organic cofactors such as flavins (Chollet et al., 1975) and a variety of other organic cofactors (R. Gee, personal communication), but no more than a trace of these compounds could be observed. The anomaly that ribulose-P2 carboxylase/oxygenase lacks a transition metal or organic cofactor could be ex- plained by the oxygenase reaction proceeding by a free radi- cal mechanism. This chapter records my studies on the mechanism of ribulose-P2 oxygenase and potential reaction mechanisms are presented and discussed. 42 43 Materials and Methods. ' Ribulose-P2 carboxylase/oxygenase from spinach leaves was purified, stored, activated and assayed as described in Chapter 1. Ribulose-P2 was synthesized and purified as de- scribed in Chapter 1. The copper chelate of lysine, Cu(lys)2, was synthesized as described by Stevens and Bush (1950). Trypsin and chymotrypsin were obtained from Millipore. Other reagents were obtained from Sigma or were of analytical reagent quality. Fluorescence measurements were made with an Aminco-Bowman Spectrophotofluorometer. Results. The Presence and Stoichiometry of Flavin in Ribulose-Pa Carboxylase/Oxygenase. Purified ribulose-P2 carboxylase/oxygenase exhibited a fluorescence excitation spectrum characteristic of a flavin (excitation maxima at 380 and 470 nm) when fluorescence emission was monitored at 525 nm (Fig. 4). The emission spectrum showed only a shoulder, with no peak resolved. Activated enzyme gave similar results with no apparent changes in fluor- escence intensity. Enzyme which was denatured with SDS (Fig. 5), digested with trypsin and chymotrypsin (data not shown) or hydrolyzed with 2.0 N HCl at 100 C (data not shown) showed excitation and emission spectra which were clearer and better resolved. A distinct emission maximum at 520 nm and excitation maxima at 380 and 450 nm are shown in Figure 4. 44 The Fluorescence Spectra of Native Ribulose-P2 Carboxylase/Oxygenase. Enzyme (13.8 mg/ml) was prepared in 0.1 M Bicine-Na+ at pH 8.0 and scanned for fluorescent excitation with emission at 520 nm and scanned for emission with excitation at 380 nm. Similar spectra were obtained with enzyme activated with 10 mM NaHCO and 10 mM MgCl 3 2° 45 . E: 56868? co_mm_Em_ .e mgzmwm E: 585662, cozozoxm Goo 0mm com Omv 00¢ 0mm 00m N— LO VIC Ansueim eoueosaionH 91010193 Figure 5. 46 The Fluorescence Spectra of Denatured Ribulose-P2 Carboxylase/Oxygenase. Enzyme (12.1 mg/ml) was denatured by heating at 100 C for 2 min in the presence of 0.1 M Bicine-Na+ (pH 8.0), 0.8% (w/v) SDS and 8% (v/v) 2-mercaptoethanol. Fluorescence scans were ob- tained for excitation with emission monitered at 525 nm and for emission with excitation at 450 nm., 47 .m mgamwm E: 56566; co_mm_Em_ E: £88.90; cozozoxm 00m 0mm 00m one cow 0mm I 1 STI‘ON Aiisueiul eouaoseiongg eniiolea Figure 6. 48 The Fluorescence Spectra of Flavin Mononucleotide. Flavin mononucleotide (10,.1M) was prepared in 0.1 M sodium formate (pH 3.2) and_scanned for fluorescence as described in Figure 2. 49 .o «gnaw; 6:. 58866; co_mm_Em_Ec £6858; cozozoxm com ‘ 0mm 00m 00m 00¢ 00¢ 0mm 00m l l in <1- ro (\l Ausueiul eoueoseiom :1 angiolaa 50 Figure 5. These spectra are very similar to those observed with authentic flavins (Fig. 6). A fluorescent compound was isolated from ribulose-P2 carboxylase/oxygenase by digestion of 50 mg of enzyme with 5% (w/w) of trypsin and chymotrypsin for 36 h at 22 C. This preparation was fractionated by chromatography on Bio-Gel P-4 which was eluted with distilled water. A fraction with fluorescence spectra similar to those in Figure 6 was recovered. This fraction was lyophilized and a sample was applied to a cellulose thin layer plate. The plate was developed with 80% (w/v) phenol:H20 in the first dimension and 2:1:l.4 (v/v/v) butanol:propionic acid:water in the second dimension. Riboflavin, flavin mononucleotide and flavin adenine dinucleotide are readily resolved by this method. The unknown fluorescent compound co-chromatographed with riboflavin under this protocol. This compound co-chro- matographed with riboflavin in 6 other solvent systems (R. Gee, personal communication). The stoichiometry of the fluorescent compound was assessed by using the internal standard method described by Koziol (1971). Ribulose-P2 carboxylase/oxygenase (12.1 mg/ml) was denatured with SDS and the relative fluorescence intensity was measured. A standard curve was generated by addition of riboflavin to the denatured enzyme and measure‘ ment of the relative fluorescent intensity. The standard curve and the fluorescence of denatured carboxylase is pre- Figure 7. 51 Quantitation of Riboflavin by Fluorescence of Ribulose-P2 Carboxylase. Enzyme (22,1M holoenzyme) was denatured with SDS as described in Figure 5 and the relative fluorescence intensity (excitation 450 nm, emission 525 nm) was determined and is indicated by the dashed line. The standard curve of ribo- flavin fluorescence was generated by addition of small aliquots of a riboflavin solution to the de- natured enzyme. The increase in fluorescence intensity is plotted as a function of the concen- tration of riboflavin added. The fluorescence of the denatured enzyme is equivalent to 0.28‘uM riboflavin. This analysis indicates that this carboxylase preparation contained 0.013 mol ribo- flavin per mol of holoenzyme. Relative Fluorescence Intensity 52 12.5 I 10.0 1 7'-5 £3.(] 22.5 [1.0 0. 0.013 mol riboflavin per mol holoenzyme 0150 0:75 1.00 [Riboflavin] pM Figure 7. 53 sented in Figure 7. The stoichiometry of riboflavin to holoenzyme is exceedingly small; these data indicate that only 0.013 mol of riboflavin were present per mol of holo- enzyme. The flavin seems to be a trace contaminant of puri- fied ribulose-P2 carboxylase/oxygenase. Attempts to Observe Intermediates in the Ribulose-P Oxygenase Reaction. Ribulose-P2 oxygenase has been pro- posed to proceed by a radical mechanism which may involve a carbon based radical derived from ribulose-P2, superoxide and 2-hydroperoxy-3 keto-pentitol-P2 (Lorimer, 1981a). The presence of these intermediates was tested by several tech- niques. Superoxide is a reactive species of oxygen generated by 2+ one electron reduction of 02. NBT will oxidize 02- and this reaction may be followed spectrophotometrically at 560 nm. Addition of NBT2+ to a ribulose-P2 oxygenase reaction did not result in dye reduction and NBT2+ did not inhibit O2 consumption in an oxygenase assay. A possible explana- tion for these results is that the active intermediate was compartmentalized from the oxidant in the enzyme catalytic site. The experiment was repeated under the following conditions which may partially denature the protein: 50 C; 0.2 M NaCNS; 0.2 M guanidine HCl; 0.8 M urea; and 1% Tri- ton-X-100. Dye reduction was not observed under any of these conditions. 54 Amino acid chelates of copper have superoxide dismutase activity and these have been used to implicate the involvement of 02- in the reaction mechanism of diamine oxidase (EC 1.4.3.6) (Younes and Weser, 1978). The copper chelate of lysine was synthesized and demonstrated to ex- hibit potent inhibition of 02- dependent cytochrome c reduc‘ tion. The copper chelate of lysine was a poor inhibitor of both ribulose-P2 carboxylase and oxygenase activities and did not inhibit either activity selectively (data not shown). Discussion. A compound which co-chromatographs with riboflavin on two dimensional chromatography and exhibits the fluorescent spectra characteristic of a flavin has been isolated from purified spinach ribulose-P2 carboxylase/oxygenase. The quantity of the compound present in the enzyme preparation was assessed by fluorescence spectroscopy using riboflavin as the standard. The stoichiometry was 0.013 mol flavin per mol holoenzyme. Therefore this compound was present at far less than 8 mol per mol of holoenzyme, which would be ex- - pected if the flavin were a required cofactor for the oxy- genase reaction. Riboflavin is probably a contaminant of the purified enzyme, which is present in trace amounts. Purified ribulose-P2 carboxylase from bean and pea leaves also binds indole-3-acetic acid with a KD of about 10.6 M 55 and stoichiometry of 1 mol indole-3-acetic acid per mol of holoenzyme (Wardrop and Poyla, 1980). The significance of flavin or auxin binding to ribulose-P2 carboxylase remains unknown. These may represent related phenomenon since the isoalloxazine ring of flavin is similar in structure to the indole ring of indole acetic acid. No evidence was obtained for a free radical mechanism 2+ for ribulose-P2 oxygenase. Neither NBT or Cu(lys)2 were effective in interrupting the oxygenase reaction. Previous 2+ investigators have reported NBT reduction by ribulose-P2 oxygenase and inhibition of O2 consumption by NBT2+ (Bhagwat and Sane, 1978b); I have been unable to repeat these re- sults. Bhagwat and Sane (1978a) also reported that super- oxide dismutase was a selective inhibitor of ribulose-P2 oxygenase. This observation could not be re-' peated in several laboratories (McCurry, 1979; Lorimer et al., 1973a). The copper chelate of lysine is a small com- pound with potent superoxide dismutase activity, and I have been unable to demonstrate selective inhibition of ribu- lose-P2 oxygenase with this reagent. All the experimental approaches utilized in these studies suffer from the handicap that unequivocal con- clusions may not be drawn. A reactive intermediate may be compartmentalized in the enzyme catalytic site, or may be too short-lived or labile to be observed by these methods. 56 The mechanism of ribulose-P2 oxygenase remains a mystery. Iron, copper and organic cofactors are not present in ribulose-P2 carboxylase/oxygenase at a stoichiometry indicative of a cofactor. In view of the absence of elect- ron delocalizing cofactors, it seems obligatory that ribulose-P2 oxygenase must proceed through a radical mechanism. Two mechanisms for ribulose-P2 oxygenase have been proposed (Lorimer, 1981a; Lorimer and Andrews, 1981). Scheme 2 shows a possible mechanism for ribulose-P2 oxygen- ase. The enediol(ate) may tautomerize to the carbanion and this species could reduce 02 by one electron, indicated by the single headed arrow. The intermediates of this reaction would be a carbon based radical and superoxide radical. These two radicals may recombine to form 2-peroxy-3- keto-pentitol-Pz. This putative intermediate of the oxygen- ase reaction may decompose by addition and elimination of hydroxyl ion. This mechanism involves the reaction of a triplet and a singlet to form two doublets, followed by reaction to form two singlets, essentially as discussed with the reduction of dioxygen by reduced flavin in the litera- ture review. Thus ribulose-P2 is proposed to react in a manner analogous to flavin. Ribulose-P2 is incapable of forming resonance structures as highly stabilized as the flavin semiquinone, although the resonance stabilization of the semidione radical is considerable (Russel and Strom, 57 .N deacom - moaoNIoiozouNm - moaoNIo... _m NW. N o 07 \e .I \e as /o: w 0 \\ _ Ioiorrmo :oiowoi/mz 101.0 _m__ _m: {m % N N N m oi \« 6.7 \m _ o 0 IO! r\l _ N ll :5 ii \ Ioloilo 0 ex” Jr :0\ /_m {m 58 . m msmcum N \m .o/ No :oi . _m 1 0\ O E .o/ \Nm 0 2') __ o ..o\ 2m .o\ /_m 59 1964; Russel, 1968) (Scheme 3). A second mechanism proposed for'ribulose-P2 oxygenase is analogous to lipid peroxidation (Scheme 4). In this mechanism the enzyme oxidizes ribulose-P2 by one electron, yielding a carbon-based radical of ribulose-P2. This could react with O2 to yield a peroxy radical intermediate. The peroxy radical may be reduced by one electron from the en- zyme yielding the same 2-peroxy-3-keto-pentital-P2 as in Scheme 2. Although this mechanism is possible in terms of the oxygen chemistry, we must consider what functional group might participate in this sort of catalysis. The mechanism involves the one electron oxidation of ribulose-P2, and con- comitant one electron reduction of an amino acid residue. Several amino acid residues, such as cysteine or tyrosine, may form radicals on oxidation; none of the amino acid side chains readily form radicals on reduction. This presents a problem to the mechanism proposed in Scheme 4. The enzyme would have to exist in the native state with an amino acid residue oxidized by one electron in a radical form to be poised for the catalytic cycle. A possible resolution of this might be the involvement of a disulfide bond in cata- lysis (Eq 3). However, no disulfides have been observed in ribulose-P2 carboxylase/oxygenase (Sugiyama et al., 1968b; Mulligan, unpublished). Thus, although either mechaniSm is possible and no data exist on which to discriminate between 60 _ + oflo I/ _ A r . _d \v Iolwlolo. J :olwlolox W 61 these mechanisms, the mechanism depicted in Scheme 4 seems less likely since one electron reduction of an amino acid residue seems chemically unreasonable. -S-S ---> -S- +5- + Ribulose-P2 ---> -S- 'S- +°Ribulose-P2 (Eq. CHAPTER 3 STIMULATION AND INHIBITION OF RIBULOSE-P2 CARBOXYLASE/OXYGENASE BY GLYOXYLATE Ribulose-P2 carboxylase/oxygenase catalyzes the first reaction of each of the two cycles of photosynthetic carbon metabolism. The ribulose-P2 carboxylase reaction fixes CO2 and initiates the reductive photosynthetic carbon cycle in which CO2 is reduced to the level of carbohydrate by a series of reactions in the chloroplasts. The ribulose-P2 oxygenase reaction consumes O2 and initiates the oxidative photosynthetic carbon cycle. This pathway pro- ceeds by a series of reactions in the peroxisomes, mito- chondria and chloroplasts and is partially responsible for the evolution of CO2 and consumption of O2 in the light. The partitioning of carbon between the reductive and oxidative photosynthetic carbon cycles is mediated by ribulose-P2 carboxylase/oxygenase. The known mechanism for modifying the flux of carbon between the cycles is by chang- ing the availability of the gaseous substrates, C02 and 02. It has been speculated that intermediates of the_oxidative photosynthetic carbon cycle may regulate the relative magni- tudes of the oxidative and reductive pathways by a feedback mechanism (Oliver and Zelitch, 1977). Oliver and Zelitch (1977) reported that glyoxylate was a potent inhibitor of CO2 fixation in isolated spinach chloroplasts, however, these authors and others (Hatch and Jensen, 1980) did not 62 63 observe inhibition of ribulose-P2 carboxylase by glyoxylate. The effect of glyoxylate on purified spinach ribulose-P2 carboxylase/oxygenase has been examined in this chapter. Materials and Methods Ribulose-P2 carboxylase/oxygenase was purified, stored, activated and assayed as described in Chapter 1. Ribulose-P2 was synthesized and purified as described in Chapter 1. CABP was prepared as previously described (Pierce et al., 1980). [14CI-glyoxylate (6.98 mCi/mmol) was obtained Sodium 1- from Amersham and solutions were lyophilized for storage when not in use. Sodium glyoxylate was obtained from Sigma, and solutions were prepared fresh daily. Other chemicals were of analytical reagent quality. Enzyme was activated for 30 min at 30 C, and glyoxylate was added to the desired concentration and incubated at 30 C. Unless otherwise indicated, the interaction of the unactivated enzyme was studied in 0.1 M Bicine-Na+ (pH 8.2), 0.2 mM EDTA and CO2 in equilibrium with the atmosphere (approximately 0.7 mM HC03-). Sodium glyoxylate was usually added at 5 or 10 mM and standard carboxylase assays were performed, except the duration of the assay was 15 s to minimize activation or inhibition during the assay. 64 Results Inhibition of Ribulose-Pa Carboxy- lase/Oxygenase by Glyoxylate. Inhibition of ribulose-P2 carboxylase by glyoxylate follows psuedo first-order kinetics with a fast initial phase and a slower second phase (Fig. 8A). Rapid inactivation was observed with the activated enzyme, and gluconate-6-P, a competitive inhibitor with respect to ribulose-P2, was exceedingly effective in protecting the enzyme from glyoxylate inhibi- tion. Bovine serum albumin at 5-fold the concentration of carboxylase was ineffective in protecting the carboxylase from inhibition. These data indicate that the interaction of glyoxylate with ribulose-P2 carboxylase was specific and that occupation of the catalytic site precluded inhibition. A double reciprocal plot of glyoxylate concentration versus the psuedo first-order rate constant for inhibition of carboxylase acitvity indicates rate saturation with a maximal rate constant for inactivation of 7 x 10.3 s-1 and K (concentration giving half the maximal rate of in- inact activation) for glyoxylate of 3.3 mM (Fig. 8B). These re- sults indicate the formation of an inhibitor4enzyme complex prior to enzyme inactivation (Meloche, 1967) (Eq. 4). E: +’]: __€’ E:.]: ___9. .£:j[.ak . (Eq. 4) Figure 8. 65 Time course of Inhibition of Ribulose-P2 Carboxylase Activity by Glyoxylate under Various Conditions. A). Time course following enzyme activity over a 4 h period with enzyme (2 mg/ml) treated under the following conditions 1) activated enzyme incubated with 1 mM gluconate-6-P prior to the addition of 1 mM glyoxylate (o); 2) activated enzyme with no glyoxylate (control) (A); 3) activated enzyme in the presence or absence of BSA (10 mg/ml) and 1 mM glyoxylate (A); 4) activated enzyme and 2 mM glyoxylate (D) b) activated enzyme and 10 mM glyoxylate,(l). Aliquots were removed at the times indicated after the addition of glyoxylate and assayed in the standard assay mixture. B). Time course over the first 3 min with enzyme (2 mg/ml) treated with 1 mM (A), 2 mM (A), 5 mM (a) and 10 mM (I) glyoxylate. The inset is a double reciprocal plot of the rate of inactivation versus glyoxylate concentration. 66 <30 1 l l l I 1 ID I ‘P ‘0 V 2.04 x oas l(mama) ION 'O.4 “0.2 O 0.2 0.4 0.6 08 [GlyoxylateI' mM-| I _|_ L e a 2 o (f - 0 <1 I (5 (4,: . I Q l O I O 4 ’- I | _1 l a a a a a 9- (bugugowei cyo) fig/moo euJ/(zu3 ' l50 l80 |20 Time (sec) Time (h) Figure 8. 67 Kinetic analysis is analogous to Michaelis-Menten where k is the observed rate of inactivation, k is inact max inact the maximal rate of inactivation and Kinact is the inhibitor concentration at 1/2 the maximal rate of inactivation (Eq. 5). km“). 2 kmaxihazi‘ ' 1 (Eq. 5) Kinac‘i‘ + I Further evidence that the enzyme forms a reversible complex with glyoxylate was indicated by the competitive in- hibition with respect to ribulose-P2 (Fig. 9A). Kinetic analysis of enzyme which was exposed to glyoxylate only dur- ing catalysis (not activation) indicated glyoxylate was essentially competitive with respect to ribulose-P2 with a KI of 2 mM. Exposure of the enzyme to glyoxylate during activation resulted in a dramatic decrease in the Vmax (Fig. 9B). This response was due to enzyme inactivation which was irreversible in the time frame of the enzyme assay. Inhibition of ribulose-P2 carboxylase and oxygenase activities was assessed after 2 h incubation of enzyme with various concentrations of glyoxylate (Fig. 10). Both carboxylase and oxygenase activities were equally inhibited and glyoxylate was not selective in inhibition of either activity. Figure 9. 68 Inhibition of Ribulose-P2 Carboxylase by Glyoxylate. Activated enzyme (2 mg/ml) was assayed for 20 s with variable ribulose-P2 (0.1 to 1.0 mM) and variable glyoxylate (0, 2.5, 10 mM).. A). Kinetic parameters obtained when glyoxylate was exposed to enzyme only during the assay. B). Kinetic parameters obtained when glyoxylate was included in the enzyme activation and assayed at the same concentration. 69 .EoLoLQTmETEENOO _oE1.on>xi I 1. m.N o.N m; D.“ w.o o.o _ _ _ — _ 0 -m 5 7 M m T. e 01m 15M, 0 N. g TIL em [2 0 A a . (q nw c: we 2. mm o 2.1 .AwdlomoSatVex Eocoaoo mile Figure 9A. 7O m.N c_o~o.aimE_...c_E.Noo _oEi.on>..r i 1.. D.N m; o; m.o 0.0 _ _ _ _ _ no t. -m x x x x N :0 a i . C n: \ mm m ] 1w r.nw.m~ 5 w. m. as [ :w i.92 no .1? . . _ _ 0. Deg mod on mm o 2.1 .leomosntvev. “Cotonou I Figure 9B. 71 Figure 10. The Inhibition of Carboxylase and Oxygenase' Activities as a Function of Glyoxylate Concentration. Activated enzyme (2 mg/ml) was incubated at each glyoxylate concentration for 2 h at 30 C. Carboxylase (O) and oxygenase (*) activities were assayed in the presence of glyoxylate at a concentration equal to that in the incubation. 72 d _ mm ow ov 35:680.. .5 5.3500 oEcho _ ON M m [.0 1, ] e t .m VJ x. om .er gr [.W l H 5 0 o 73 Figure 11. The Reversibility of Glyoxylate Inhibition. Enzyme (10 mg/ml) was treated with 10 mM glyoxylate for 3 h at 30 C. Aliquots (10,11) were taken and diluted (1:100) into 0.99 ml of assay buffer. Recovered enzyme activity was determined at timed intervals after dilution by initiating the reactions with ribulose-P2. Control assays were run in the presence of 0.1 mM glyoxylate (equivalent to glyOxylate carried over with enzyme into the assay). In (75 activity) 4 .00 4.50 4.25 3.75 74 c3:3 .50 r 2 time (min) Hgme H. 75 The kinetics of dissociation of glyoxylate from the en- zyme complex was assessed by inhibition of carboxylase with 10 mM glyoxylate for 3 hours, 100 fold dilution of the en- zyme into inhibitor-free activation buffer and assay of car- boxylase activity with time (Fig. 11). Reactivation follow- ed psuedo first-order kinetics with a rate constant for reactivation of 1.2 X 10.2 s“1 and corresponding half-life of 60 5. Since reactivation was slow compared to the time frame of an enzyme assay (15-30 s), glyoxylate has been treated kinetically as an irreversible inhibitor. Figure 11 demonstrates that glyoxylate is actually a slowly reversible inhibitor. The Stimulation of the Unactivated Enzyme by Glyoxylate. Addition of glyoxylate to unactivated carboxylase initially stimulated carboxylase activity which was followed by time-dependent inactivation (Fig. 12). The stimulation of activity could be observed if 1,3 or 10 mM NaHCO3 were in- cluded prior to glyoxylate addition. Some stimulation of carboxylase activity could also be observed if 20 mM MgCl2 was included prior to glyoxylate addition. If HCO3- and MgCl2 were added together to activate the enzyme, only time dependent inhibition was observed. Addition of glycolate to the unactivated enzyme did not cause any change in the carboxylase acitvity, indicating that this was not a nonspecific response to an organic acid. Enzyme activated 76 Figure 12. Stimulation of Ribulose-P2 Carboxylase Activity By Glyoxylate. The indicated concentration of glyoxylate was added to a carboxylase solution (2 mg/ml) in 0.1 M Bicine-Na+ (pH 8.2), 0.2 mM EDTA and air levels of C02. Carboxylase activity was determined by assay of 10 ”l aliquots at the indicated times. Glyoxylate concentrations are as follows; 0, 0 mM; A, 5 mM; X, 10 mM; *, 20 mM; and 2,-5mM glyoxylate with 20 mM MgC12. 77 1-0 L 0.8 L 0.6 L .4 l 0 nmol CO2 min" mg"protein 0.2 - . . . 0.0 I T r I I I I 0 15 30 45 60 75 90 time after the addition of glyoxylate (seconds) Figure l2. 78 with 3.3 mM MgC12, MnCl2 or CoCl2 showed similar glyoxy- late-dependent carboxylase stimulation. The inhibition phase which occurred after addition of glyoxylate to the un- activated enzyme exhibited biphasicity with psuedo first-or- ‘1 and 8 x 10'5 5'1. der rate constants of 1 X 10-3 s The interaction between unactivated enzyme and glyoxyl- ate was compared with the interaction with the effector, gluconate-G-P. Unactivated enzyme was exposed to 1 mM glu- conate-6-P or 10 mM glyoxylate and carboxylase activity was measured over time (Fig. 13). The interactions of glyoxy- late and gluconate-G-P with the enzyme were dissimilar. Glyoxylate elicted a rapid increase in carboxylase activity in the presence or absence of MgClz; gluconate-S-P treatment slightly inhibited carboxylase activity in the absence of MgCl2 and resulted in a slow activation of the activity in the presence of MgClz. The enzyme activity after 1 h was 1 ‘mg-1 protein for carboxylase incubated 0.085 pmol'min- with gluconate-6-P and 1.30 for carboxylase treated with gluconate-G-P and 20 mM MgC12. The interaction of glyoxylate with unactivated enzyme was probed by examination of the binding of C02, CABP and glyoxylate (Table 2). Glyoxylate did not stimulate or in- hibit CO2 or CABP incorporation into the quaternary complex 2+-CABP. In addition, CABP was ineffective of enzyme-COZ-Mg in trapping glyoxylate beyond that bound in the absence of CABP. Gluconate-G-P stimulated a large increase in bound 79 Figure 13. The Effect of Gluconate-é-P and Glyoxylate on Unactivated Ribulose-P2 Carboxylase. In this experiment, CO2 depleted reagents were prepared by using freshly boiled water and gassing all solutions with N2 during handling and for storage. Glyoxylate (10 mM) (0) or 6-P-gluconate (1 mM) (*) was added to 2 mg/ml ribulose-P2 carboxylase in 0.1 M Bicine-Na+ (pH 8.2) and 0.2 mM EDTA with or without 20 mM MgClZ. Carboxylase activity was assayed with zo‘pg of protein at the indicated times. Solid lines are treatments in the absence of MgClz. Dashed lines are for treatments which contained 20 mM MgC12. 80 ('0 Cd C: -45 ———— \ ’ p \‘\\ ” / .5; / "Er' / a) / +1 / / e / ’x\\ / l / ’ tn“! 7———-*’ 4 ED / 'I' .E a I» O O... '60- E 3. + O o I I I 0 15 30 - 45 time after addition (seconds) Figure 13. Table 2. Binding of CO , Activated Ribalose- 81 CABP and Glyoxylate by Partially P2 Carboxylase. Ribulose-P2 carboxylase (1 mg protein, 14.55 nmol protomer) was incubated with 0.1 M Bicine-Na+ (pH 8.2), 0.2 mM EDTA and 1 mM NaHCO Sodium glyoxylate (10 mM), MgCl (20 mM) were 5, 4 nmol of CABP to a concentration were incubated for diluted 10 fold in gluconate-G-B (1 mM) and added where indicated. After 45 was added, and MgCl was added of 20 mM. The protgin solutions 45 min, and the radioisotope was radiospecific activity. The loosely bound ligands were allowed to exchange for 30 min (5 h for CABP binding). The protein was then precipitated with 20% polyethylene glygol 4000 and 20 mM MgC prepared in 50 mM Bicine-Na (pH 1 8.2) and 0.1 mg EDTA. The precipitate was collect- ed by centrifugation (20 min, 48,000 x g) and washed twice by suspension in the polyethylene glycol and MgCl2 media and centrifugation. The protein was dissolved in 50 mM Bicine-Na+ (pH 8.2), 0.1 mM EDTA and 5 mM NaHCO for scintillation counting. The radioactively labelled species is indicated (species bound) for each determination. Glyoxylate is abbreviated as Glx. Additions to Trapping Radiolabelled mol bound Activation Condition Species Bound per mol protomer 10 mM Glx CABP,M92+ co2 0.11 none CABP,M92+ coz 0.13 10 mM Glx CABP,M92+ CABP 0.11 none CABP,M92+ CABP 0.10 10 mM Glx CABP,M92+ Glx 0.34 10 mM Glx none Glx 0.32 10 mM Glx 2+ 20 mM MgCl2 CABP,Mg co2 0.38 20 mM MgCl2 CABP,M92+ coz 0.35 20 mM MgCl2 and 1 mM 2+ 6-P-gluconate CABP,Mg C02 1.08 82 CO2 due to carbamate formation. Glyoxylate interacts with ribulose-P2 carboxylase in a manner which is unlike the effector gluconate-G-P and apparently will not substitute for carbamate formation as judged by CABP binding or CABP trapping of glyoxylate. Discussion Glyoxylate is a slowly reversible inhibitor of ribu- lose-P2 carboxylase/oxygenase. The fully activated enzyme is inhibited by glyoxylate at about an order of magnitude faster than the unactivated form. Activation by C02 and 2+ does result in conformational changes in the protein M9 and this could alter the accessibility or reactivity of an amino acid residue. The interaction of ribulose-P2 carboxy- lase with glyoxylate appears to be a specific interaction based on several criteria: the presence of excess bovine serum albumin offered no protection against glyoxylate in- hibition; gluconate-6-P, a competitive inhibitor with re- spect to ribulose-P2, protected carboxylase activity from inhibition by glyoxylate; glyoxylate was a competitive in- hibitor with respect to ribulose-P2; inactivation was psuedo first-order with rate saturation; and complete inactivation corresponded to 1 mol of glyoxylate per mol of protomer when incorporation was corrected for nonspecific incorporation (Cook, personal communication). The interactions of glyoxy- late with ribulose-P2 carboxylase satisfy the criteria which 83 Hartman et al. (1978) proposed to establish a compound as an affinity label. Glyoxylate was irreversibly incorporated into the pro- tein after reduction with NaBH4 and SDS gel electrophoreses l4C-glyoxylate demon- of the protein derivitized with.l- strated that the large subunit was labelled (Cook, unpublished observation). In addition, correlation of en- zyme inhibition and glyoxylate incorporation indicated that complete loss of enzyme activity occurred with derivitiza- tion of a single lysyl residue per protomer. These data indicate that glyoxylate forms a Schiff-base with a lysyl residue which is probably in the region of the catalytic site. This hypothesis has recently been confirmed by amino acid sequencing of the derivitized peptide after proteolysis (C.M. Cook, F.C. Hartman and N.E. Tolbert, personal communi- cation). Lysyl residue 175 was covalently modified by glyoxylate after reduction with NaBH4. This residue has been derivitized by pyridoxal-P (Spellman et al., 1979) and various affinity ligands (Schloss et al., 1978b). This lysyl residue exhibits enhanced nucleophilicity toward car- bonyl containing reagents and reactivity toward active site probes. The reactive nature of this residue may indicate that it has some catalytic function such as proton abstrac- tion during the enolization of ribulose-P2. The biphasic nature of glyoxylate inhibition was un- expected. Although the incorporation of glyoxylate 84 correlated with the observed inhibition of activity, the reason for the change in the rate of inactivation was not evident. The extent of incorporation which occurred before the change in rate varied with the glyoxylate concentration. The biphasic rates of inhibition could be due to: cooperativity between subunits (e.g. negative allosterism); 2 distinct residues which are derivitized; or 2 conformers of the enzyme which show different rates of inactivation with glyoxylate. Since lysyl 175 was specifically derivitized, it seems unlikely that more than one amino acid residue was involved. The enzyme exhibits Michaelis-Menten kinetics, and cooperativity between subunits with substrate binding or catalysis has not been observed. However, it is possible that the subunits act cooperatively with inhibition by glyoxylate. Different forms of the activated enzyme may also exist, however there is no indication of this from the literature. Glyoxylate caused a small stimulation of the activity of the unactivated protein. The mechanism responsible for this stimulation is not known, although glyoxylate behaved in a manner clearly unlike an effector, gluconate-G-P. Per- haps glyoxylate interacted with the unactivated protein in a manner that simulates the molecular changes of activation. One hypothesis proposed during this investigation was that glyoxylate formed a Schiff-base with lysyl 201, the residue that reacts with CO2 to form the lysyl-carbamate during 85 activation. The glyoxylate adduct of lysyl 201 might be an analogue of the carbamate, and consequently glyoxylate might alleviate the CO2 requirement of activation. This sort of interaction could explain the results, but one would predict that glyoxylate should stimulate CABP incorporation into the tight binding complex and this was not observed. The mechanism of glyoxylate stimulation of the unactivated en- zyme is unknown. Glyoxylate did not act as an effector in stimulation of carbamate formation. Glyoxylate did not stimulate CABP incorporation into a tight-binding complex with the enzyme, which might be indicative of Schiff-base formation with lysyl 201. . Glyoxylate has been demonstrated to be a slowly re- versible inhibitor or ribulose-P2 carboxylase. The inhibi- tion was not selective against the carboxylase or oxygenase activities. The inhibition exhibited by glyoxylate was due to Schiff-base formation with lysyl 175, a reactive cata- lytic site amino acid residue. Ribulose-P2 carboxylase is weakly sensitive to glyoxylate as a competitive inhibitor (KI = 2 mM) or as an affinity label (K = 3.3 mM). inact Since glyoxylate is a peroxisomal metabolite and glyoxylate reductase (NADP+) activity exists in the chloroplast (Chapter 4), glyoxylate should not accumulate in the chloro- plast. These'facts, together with the lack of selective in- hibition of ribulose-P2 carboxylase and oxygenase activi- ties, indicate that glyoxylate does not serve any physio- 86 logical role in the regulation of photosynthesis or photore- spiration as mediated by ribulose-P2 carboxylase/oxygenase. Acknowledgement This chapter represents my contribution to a collabora- tion with Cathy M. Cook. Figures 8 and 10 represent experi- ments performed by Cathy M. Cook. They have been included in the thesis for clarity and I wish to thank her for allow- ing me to use them. CHAPTER 4 THE EFFECT OF GLYOXYLATE ON PHOTOSYNTHESIS BY INTACT CHLOROPLASTS The partitioning of carbon between the reductive and oxidative photosynthetic carbon cycles is mediated by ribulose-P2 carboxylase/- oxygenase. The known mechanism for modifying the flux of carbon between the cycles is by changing the availability of the gaseous substrates, C02 and 02. It has been speculated that intermediates of the oxidative photosynthetic carbon cycle may regulate the relative magnitudes of the oxidative and reductive pathways by a feedback mechanism (Oliver and Zelitch, 1977; Oliver, 1980). Oliver and Zelitch (1977) reported that glyoxylate was a potent inhibitor of 002 fixation in isolated spinach chloroplasts, but that glyoxylate did not inhibit ribulose-P2 carboxylase activity. Glyoxylate is an inhibitor of purified ribulose-P2 carboxylase/oxygenase jg_yit£g (Chapter 3; Cook and Tolbert, 1982). Glyoxylate is a competitive inhibitor with respect to ribulose-P2, and in addition forms an adduct with an amino acid residue of ribulose-P2 carboxylase/oxygenase. Glyoxylate does not selectively inhibit the carboxylase or oxygenase activities. The inhibitory nature of glyoxylate suggested that it might exert some regulatory effect on photosynthesis. In view of these possibilities we 87 88 have examined the effect of glyoxylate on photosynthesis by intact chlorOplasts. Materials and Methods Chloroplasts. Intact chlor0plasts were isolated by the procedure of Nakatani and Barber (1977) from spinach leaves (§pjnacia oleracea L.) which were obtained at a local market. Chloroplasts were suspended in a photosynthetic media composed of 330 mM sorbitol, 20 mM Bicine, 2 mM EDTA, 1 mM MgCl2, 1 mM MnClz and 50 mM sodium pyrophosphate which was adjusted to pH 8.0 with NaOH. The intactness of the chloro- plast preparations was typically 70% as determined by the ferricyanide permeability procedure (Nakatani and Barber, 1977). Chlorophyll was determined spectrophotometrically in 80% acetone (Arnon, 1949). Photosynthetic C07 Fixation. An aliquot of chlorOplasts was suspended in photosynthetic media containing 1 WM ribose-S-P, 0.5 mM ADP and 5 mM NaHC03. Photosynthetic C02 fixation was stimulated by the addition of ribose-S-P and ADP by 10-15%, but C02 fixation was not dependent on these compounds. The chloroplasts (approximately 100 ug Chl-ml‘l) were placed in a flattened test tube in a glass waterbath at 22°C. Illumination from a slide projector was filtered through a solution of CuSO4. Photosynthetically active radiation was measured with a Lambda Instruments L1-185 quantum meter as 2500 uEinsteins-s'l'm'z. Photosynthesis was determined by the rate of 14C02 fixation and was typically 50 nmol-mg'IChl-h'l. 89 Assay of Ribulose-Pp Carboxylase from Intact ChlorOplasts. Assays were performed essentially as described by Bahr and Jensen (1978). A 0.025 ml aliquot of chloroplasts (2.5 pg Chl) in photosynthetic media was added to 0.225 ml of the carboxylase assay media. The assay media contained 27.5 mM Bicine-Na+ (pH 8.2), 22 mM MgClz, 10 mM NaH14co3 (30.4 uCi-umol‘l), 1.1 mM dithiothreitol, and 0.55 mM ribulose-P2. The carboxylase assay was incubated for 30 s at 22°C and terminated by the addition of 0.1 ml of 2 N HCl. The assay mixtures were dried at 95°C and analyzed for 14C02 incorporation into acid stable products by scintillation counting. The specific radioactivity was corrected to reflect the dilution of the isotope with unlabeled NaHC03 which was added with the chlor0plast preparation. An enzyme unit was defined as the amount of enzyme which catalyzed the formation of 1 nmol of acid stable 14C in 1 min. Ribulose-P; Determination. This procedure was similar to that described by Sicher et al. (1979). Total ribulose-P2 was determined by addition of a 0.1 ml aliquot of chloroplasts (7 to 10 ug Chl) to 0.9 ml of reaction media containing 27.5 mM Bicine-Na+ (pH 8.2), 22 mM MgCl2, 11 mM NaH14co3 (3_o.6 uCi-umol‘l), 172 ug-ml'1 of purified spinach ribulose-P2 carboxylase/oxygenase, 0.55 mM 2,6-dichlorophenol-indophenol and 0.44 mM carbonyl cyanide m—chlorophenylhydrazone. Ribulose-Pz which was not in the chloroplast was determined by centrifugation (15 5, 11,000 x g) of the chloroplast suspension and determination of ribulose-P2 in 0.1 ml of the supernatant solution. Determinations were terminated after 30 min with 0.2 ml of 2 N HCl and counted for 14C incorporation as described above. ChlorOplast ribulose-P2 was calculated by 90 correcting the total ribulose-P2 by the amount observed in the supernatant solution. The amount of ribulose-P2 in the supernatant solution was usually less than 10% of the total ribulose-P2. Glyoxylate Reductase (NADPT). This reductase was assayed in 50 "M potassium phosphate buffer (pH 6.2) and 0.17 ”M NADPH. The rate of change in absorbance was measured at 340 nm and the enzymatic rate was calculated with the molar extinction coefficient for NADPH. Paper Chromatographic Separation of Metabolites. Photosynthesis by chloroplasts was terminated by addition of formic acid to a final concentration of 0.67 N. The suspensions were diluted 10 fold with water, and centrifuged (10 min, 27,000 x g). The supernatant solutions were decanted and the volumes were reduced with a Buchler vortex-evaporator. Equivalent radioactivity was applied to each chromatogram (Nhatman 4 paper). The chromatograms were developed by descending chromatography with 80% phenol in water (w/v) in the first dimension and with n-butanolzpropionic acid:H20 (2:1:1.4 by volume) in the second dimension. The chromatograms were dried briefly after chromatography in the second dimension and sprayed with 0.5 M NaHC03 to minimize loss of volatile acids. The location of radioactive metabolites was assessed by exposure of the chromatogram to x-ray film. Fructose-P7 Determination. A chloroplast preparation (5 ml, 100 ug Chl'ml‘l) was allowed to photosynthesize for 10 min and then glyoxylate or formate was added to a concentration of 10 mM (equivalent volume of water added for the control). Photosynthesis proceeded for another 10 min and then the suspension was acidified with 2 ml of 2 N HCl. The suspension was centrifuged and an aliquot of D-[U-14CJ-fructose-P2-(l uCi, 239 uCi°umol'1) was added. 91 The fructose-P2 was purified by chromatography on Dowex l-Cl with a linear 0 to 0.5 M LiCl gradient prepared in 5 mM HCl. Fractions of the fructose-P2 peak were pooled and the phosphate ester was precipitated as the barium salt in the presence of 50% ethanol (v/v). The barium salt of fructose-P2 was dissolved by treatment with Dowex SO-H+ and neutralized with l M triethanolamine. The amount of fructose-P2 was enzymatically determined by conversion to glycerol-P. The change in absorbance at 340 nm was recorded after adding aldolase to a solution of fructose-P2 with NADH, triose-P isomerase and glycerol-P dehydrogenase. Materials. Ribulose-Pz was synthesized and purified as described in Chapter 1. Ribulose-Pz carboxylase/oxygenase was purified from spinach leaves and stored as described in Chapter 1. Sodium glyoxylate was obtained from Sigma and solutions were made fresh daily. Sodium 1-[14CJ-glyoxylate (6.98 mCi°mmol'1) was obtained from Amersham and solutions were freeze-dried for storage when not in use. Paper chromatography of the 1-[14CJ-glyoxylate preparation indicated that it contained less than 1% [14CJ-glycolate. Results Activity and Activation of Ribulosegfiz Carboxylase in Intact Chloroplasts. Glyoxylate at 10 mM inhibited C02 fixation by intact chloroplasts about 30%, and confirmed the results of Oliver and Zelitch (1977). Since ribulose-P2 carboxylase/oxygenase is inhibited 92 by glyoxylate in 11359, the possibility that this mechanism accounted for the inhibition of C02 fixation was investigated. However the activation kinetics of ribulose-P2 carboxylase in intact spinach chloroplasts during illumination or its activity upon chloroplast lysis did not change significantly with 10 mM of glyoxylate (Fig. 14). Typically 1 to 4 fold activation of the enzyme was observed after the chloroplasts were transferred into the light in the presence of 10 mM NaHC03. Frequently, there was a slight lag of about 1 min in the activation of ribulose-P2 carboxylase when glyoxylate was included. When chlor0plasts were incubated with 10 mM glyoxylate for 15 min in the light or dark without NaHC03 and then transferred into the light with the addition of NaHC03, substantial inhibition of ribulose-P2 carboxylase activity was observed in the dark pre-incubated condition (Fig. 15). This was consistent with lg 113:9 inhibition of ribulose-P2 carboxylase by glyoxylate. However in the light there was a smaller effect of exogenous glyoxylate on ribulose-P2 carboxylase activity. One explanation of the differential effect might be due to glyoxylate reduction by the chlor0plast (see next section). Ribulose-Pz levels in illuminated chloroplasts were measured with or without glyoxylate (Fig. 16). The amount of ribulose-P2 increased approximately two fold in the control, similar to results °obtained by Sicher et al. (1979). However, in the chloroplast sample treated with 10 mM glyoxylate, the ribulose-P2 level declined by about 40%. When the experiment in Figure 16 was repeated without addition of ribose-S-P and ADP, the initial level of ribulose-P2 was 12 nmol-mg'1 Chl in the presence or absence of glyoxylate and then changed similarly to the data in Figure 16. These data indicate 93 Figure 14. The Effect of Glyoxylate on Ribulose-Pz Carboxylase Activity in Chloroplasts. ChlorOplasts were added to photosynthetic media (pH 8.0, 100 ug Chl-ml'l, 10 mM NaHC03, with or without 10 mM glyoxylate) and illuminated. Aliquots were withdrawn and assayed for ribulose-P2 carboxylase activity. 94 m5 aE\o£c3 _ u £52 82.9888 $1.832 Time (min) 4 Figure l4. 95 Figure 15. The Effect of Preincubation with Glyoxylate and Illumination on Ribulose-Pz Carboxylase Activity in ChlorOplasts. Chloroplasts (100 ug Chl-ml'1 in photosynthetic media, pH 8.0) were incubated for 15 min at 25°C with or without illumination and 10 mM glyoxylate. The suspensions were then illuminated and NaHC03 added to a final concentration of 10 mM. Aliquots were withdrawn and assayed for ribulose-P2 carboxylase activity. Ribulose—P2 Carboxylase Actuvnty (units/ mg Chl) 2 1 l 96 7* I T r r l 4 s Time (min) Figure l5. 97 Figure 16. The Effect of Glyoxylate on Ribulose-P2 Levels in Chlor0plasts. Chlor0plasts were suspended in photosynthetic media as described in the legend of Fig. 1 and illuminated with or without 10 mM glyoxylate. Aliquots were withdrawn and analyzed for chlor0plast ribulose-P2 as described in the methods. Similar results were obtained with or without ribose-S-P and ADP in the photosynthetic media. 2 Chloroplast Ribulose—P (nMoI/mg Chl) 98 D 9. 00°— Control a o-l (D D <- JFNu ’0 mM s- ‘° ------ ‘3‘. ”we: ~-1»-__~~ ~~~~~~ O T T T I r °o 2 4 6 . e 10 Time (min) Figure l6, 99 that the activity of ribulose-P2 carboxylase was not substantially inhibited in the light by glyoxylate, but rather the regeneration of ribulose-P2 may have been inhibited by glyoxylate, consequently reducing the rate of C02 fixation in intact chlor0plasts. glyoxylate Metabolism in Chloroplasts. Intact chloroplasts (104 ug Chl-ml'l) were incubated with 0.57 mM 1-[14CJ-glyoxylate (2 uCi) for 20 min in photosynthetic media at pH 6 with or without illumination. Two-dimensional chromatography indicated the partial reduction of glyoxylate to glycolate by intact chloroplast preparations as has been observed previously by Kearney and Talbert (1962) with chloroplasts prepared with concentrated salt solutions and Asada et al. (1965) with a reconstituted system. In the illuminated chloroplast preparation, 67% of the radioactivity was recovered as glycolate. This rate of glyoxylate reduction is equivalent to 11 nmol-mg"1 Chl-h‘l. When 1.0 mM NaHC03 was added to the chloroplast preparation for photosynthetic utilization of NADPH, similar results were obtained with 68% of the radioactivity recovered as glycolate. The chlor0plast preparation which was not illuminated also reduced 40% of the glyoxylate to glycolate in 20 min. Reduction of glyoxylate by illuminated chloroplasts was also observed at pH 8. Only a trace (2% or less) of the radioactivity was found in glycine in these experiments. Glyoxylate reductase (NADP+) was present in the intact chloroplast preparations. This enzyme has previously been localized in spinach chlor0plasts by iSOpycnic sucrose gradient centrifugation (Talbert et al., 1970). The enzyme activity was completely soluble after chloroplast lysis by osmotic shock. No activity remained with 100 the membrane pellet after lysis and centrifugation. Kinetic studies with variable glyoxylate concentrations indicated a maximal rate of glyoxylate reduction of about 3 pmol-mg'1 Chl-h'1 and an apparent Km(glyoxylate) of 140 pM. Substrate inhibition occurred at high glyoxylate concentrations (2_500 pM). The rate of glyoxylate reduction observed spectrophotometrically (3 pmol-mg’IChl-h'l) was somewhat less than the rate of glyoxylate reduction observed radiometrically (11 nmol -mg'1 Chl°h‘1). Since glyoxylate reduction is presumably coupled to NADPH in the chloroplast, glyoxylate dependent 02 evolution should occur in a manner similar to C02 or glycerate-B-P dependent 02 evolution. However no glyoxylate dependent 02 evolution was observed when intact chloroplast preparations were illuminated, perhaps because of the low activity of glyoxylate reductase. The Effects of Salts of Weak Acids on Photosynthesis. Glyoxylate (10 mM) inhibited C02 fixation by intact chlorOplasts about 30% when the pH of the incubation media was high (pH 8.0). Lactate, acetate and glycolate at 10 mM also inhibited C02 fixation under these conditions by 7, 9 and 19%, respectively. In addition formate was similarly inhibitory. Even sodium bicarbonate at 20 mM was inhibitory toward photosynthesis under these conditions, which Enser and Heber (1980) attributed to the uptake of C02, which resulted in the formation of the weak acid, carbonic acid. If the pH of the media was decreased to 6.0, these weak acids were much more inhibitory. The concentration of glyoxylate and glycolate which inhibited C02 fixation by 50% in intact chloroplasts at pH 6.0 were 50 and 150 pM, respectively (Fig. 17). The inhibition of photosynthesis by salts of these weak acids and the pH 101 Figure 17. The Effect of Glyoxylate and Glycolate on Photosynthesis by Intact ChlorOplasts. Chloroplasts were suspended in photosynthetic media (pH 6.0, 1 mM NaHC03, 100 pg Chl'ml’l) which contained various concentrations of glyoxylate and glycolate. The rate of photosynthesis was determined over the linear portion of the 14C02 fixation time course. l02 I I T -4 -a -2 -1 I -5 Log of the Glyoxylate or Glycolate Molarity oo.u d — d J Omec omea Dveo DN.O 8:2: N8 to 33. east”. 8 no.0 Figure l7. 103 sensitivity toward inhibition suggested that these acids inhibited photosynthesis by a common mechanism, possibly by entering the chlorOplast as the protonated acid and acidifying the stroma. Effect of Glyoxylate Inhibition on the Products of Photosynthetic C02Fixation. To determine whether some enzymatic step was inhibited by glyoxylate, intact chloroplasts were provided with 14C02 for 10 min to label the metabolites and then exposed to 10 mM glyoxylate in the light for an additional 10 min with the 14c02. The labeled products were separated by two-dimensional paper chromatography. Several compounds which migrated at the RF of sugar bisphosphates contained a higher percentage of the 14C when the chloroplasts were incubated with glyoxylate than in the untreated controls. The level of fructose-P2 was measured enzymatically by coupling with aldolase and measuring NADH oxidation by glycerol-P dehydrogenase (see methods). After 10 min of photosynthesis, the concentration of fructose-P2 was 70 nmol-mg'1 Chl in the control. When the chlorOplasts were exposed to 10 mM glyoxylate or formate during photosynthesis, the level of fructose-P2 increased to 200 or 190 nmol-mg'1 Chl, respectively. Discussion Glyoxylate inhibited photosynthesis in intact chloroplasts, as observed by Oliver and Zelitch (1977). The inhibition was not specific for glyoxylate, since salts of other weak acids such as lactate, glyco- late, acetate and formate also inhibited photosynthesis. Also the inhibition of photosynthesis by glyoxylate was more severe at pH 6 than 104 at 8. The non-specific inhibition and pH dependence suggest that the mechanism of inhibition by the salts of these weak acids was in part the result of the permeability of the acid form to the chloroplast envelope and ionization in the alkaline environment of the stroma. The acid would equilibrate across the chloroplast envelOpe and the concentration of anions would distribute counter to the hydrogen ion gradient. Therefore the anion would accumulate in the alkaline compartment, resulting in a decrease of the pH of the compartment and concentration of the anion above the solution concentration. Acidification of the chloroplast stroma by 0.32 pH units was detected in the presence of 10 mM formate by Enser and Heber (1980). The level of ribulose-P2 in the chloroplast dropped after illumination in the presence of glyoxylate in contrast to the rise in ribulose-P2 levels observed in the control. Thus the regeneration of ribulose-P2 may be inhibited by glyoxylate, and chromatographic analysis indicated that fructose-P2 accumulated. Increased fructose-P2 levels in response to glyoxylate or formate treatment of intact chlorOplasts has also been observed by others (Enser and Heber, 1980; Lawyer et al., 1983). In our experiments, fructose-P2 levels were over 2-fold higher when photosynthesis in intact chloroplasts was inhibited by either glyoxylate or formate. These results suggested that fructose-P2 phosphatase was a site of inhibition, but that this effect was not a specific response to glyoxylate. The activity of fructose-P2 phosphatase is strongly effected by the pH and Mg++ concentration of its environment. Laing et al. (1981) reported that fructose-P2 phosphatase activity decreased about 50% with a pH shift from 7.9 to 7.6. 105 Fructose-P2 phosphatase is also activated by thioredoxin-dependent reduction (Buchanan, 1980) and inhibited by H202 oxidation of the sensitive sulfhydryl groups of the enzyme (Charles and Halliwell, 1981). Glyoxylate is known to form adducts with free sulfhydryl groups and free amines (Hamilton et al., 1979), and might also inhibit fructose-P2 phosphatase directly or by interfering with thioredoxin-dependent activation. However the effect of salts of other weak acids on C02 fixation and fructose-P2 levels suggests that the major inhibition is not related to the reactivity of carbon 2 glyoxylate, but rather to stromal acidification or anion accumulation. Since glyoxylate is an inhibitor of ribulose-P2 carboxylase/oxygenase in yitrg, it was expected that inhibition would be observed in the intact chloroplast system. Glyoxylate inhibits isolated ribulose-P2 carboxylase/oxygenase by two mechanisms. Glyoxylate is a competitive inhibitor with a KI of about 2 mM, and in addition reacts slowly with an amino acid residue of ribulose-P2 carboxylase/oxygenase to fonn an adduct (Chapter 3). Adduct formation results in severe inhibition. Under the conditions of the carboxylase assays performed in this paper, ribulose-P2 was present at 20 times Km and glyoxylate was present at 0.5 times Ki° Therefore only about 1% inhibition of ribulose-P2 carboxylase could be expected due to glyoxylate as a competitive inhibitor in the assay. Inhibition of ribulose-P2 carboxylase activity due to covalent adduct formation was tested under the condition of the assay, and was not substantial, except perhaps in the experiment in which chloroplasts were preincu- bated with 10 mM glyoxylate for 15 minutes in the dark. 106 The intact chloroplast preparations contained glyoxylate reductase (NADPT) activity which was readily observed by spectrophotometric or radiometric assays. This enzyme has been partially purified and characterized from tobacco and spinach leaves (Talbert et al., 1970; Zelitch and Gotto, 1962). Glyoxylate might inhibit photosynthesis by depletion of the NADPH pool by the activity of glyoxylate reductase. The rate of glyoxylate reduction was maximally at 20% of the rate of C02 fixation, which could account for the inhibition of photosynthesis. The role of glyoxylate reductase is unclear. There is still no clear evidence for a glycolate-glyoxylate shuttle. Perhaps the enzyme is responsible for the removal of glyoxylate from the chloroplast by reduction followed by excretion of glycolate. It may thus serve as a means of removing a reactive compound which may inhibit ribulose-P2 carboxylase/oxygenase or some other enzyme required in chlor0plast metabolism. In our intact chloroplast system, ribulose-P2 levels (measured enzymatically) decreased in response to glyoxylate treatment, in contrast to a control in which the ribulose-P2 level doubled. However, Lawyer et al. (1983) observed increased 14C labelling in ribulose-P2 in intact chloroplasts after exposure to glyoxylate. Glyoxylate was approximately 50% inhibitory toward C02 fixation under their conditions (5 mM glyoxylate, pH 7.6), but only 30% inhibition was observed under our conditions (10 mM glyoxylate, pH 8.0). It is possible that little inhibition of ribulose-P2 carboxylase occurs when glyoxylate is partially inhibitory to photosynthesis, but when glyoxylate inhibition is more severe, substantial carboxylase inhibition does occur. In addition, the photosynthetic media used by 107 the two groups were somewhat different. Our photosynthetic media contained pyrophosphate, which is slowly hydrolysed (Walker, 1971), in contrast to the media used by Lawyer et al. which contained 0.5 mM P1. Thus the two chloroplast preparations may have had different metabolic conditions, since Pi is known to have dramatic effects on chloroplast metabolism. The mechanism of glyoxylate inhibition of photosynthesis might involve stromal acidification, NADPH depletion or carboxylase inactivation. Our data indicate that carboxylase inactivation was not substantial. NADPH depletion due to glyoxylate reduction is a possible factor. The nonspecific response of photosynthesis to salts of weak acids suggests that stromal acidification probably was a factor. Regardless of the mechanism of glyoxylate inhibition of photosynthesis, it seem unlikely that glyoxylate serves any regulatory role in viva, since glyoxylate synthesis is compartmentalized in the peroxisome and this metabolite should not accumulate in the chlor0plast. CHAPTER 5 THE EFFECT OF TRYPSIN ON RIBULOSE’P CARBOXYLASE/OXYGENASE. 2 I became interested in correlating the kinetic para- meters for the carboxylase reaction because of the apparent relationship between Km (C02) and the type of photosynthetic carbon metabolism. The carboxylase from C4 plants and other organisms that accumulate CO2 tend to have a greater Km (C02) than the enzyme from C3 plants (Jordan and Ogren, 1981; Yeoh et al., 1980, 1981; Bird et al., 1982; Badger, 1980). Energy is used in the formation of the Michaelis complex and tighter binding results in a decreased Km. How- ever, the energy used in binding must be overcome to attain the transition state. Therefore, high affinity of an enzyme for a substrate should result in a low Km, a low kcat and a high activation energy (Fersht, 1977). Therefore it is ex- pected that an enzyme would tend to bind substrate weakly or moderately and consequently minimize the energy required to attain the transition state. Maximizing k /Km optimizes cat the use of energy in catalysis and Albery and Knowles (1976) have proposed that enzymes have evolved toward maximization of kcat/Km based on detailed kinetic studies of triose-P isomerase. These concepts lead to interesting consequences when they are applied to ribulose-P2 carboxylase in plants that accumulate CO2 and those plants that do not. C3 plants 108 109 have no known mechanism for the accumulation of C02. The only mechanism which is available to create a gradient of CO2 for diffusion into the leaf is by removing CO2 by fixa- tion with ribulose-P2 carboxylase. Therefore, evolution might result in a carboxylase with a low Km(C02) to minimize the steady-state concentration of CO2 and maximize diffu- sion. Indeed, the Km (C02) of ribulose-P2 carboxylase from C3 plants tends to be small. However, the penalty paid for a low Km is a commensurate increase in the energy required to attain the transition state. Thus C3 plant carboxylases may have evolved toward a reduced Km (C02) and reduced kcat’ A C3 plant might partially compensate for a low kcat of the carboxylase by increased enzyme concentration. C4 plants have a mechanism to concentrate CO2 and the C4 plant ribulose-P2 carboxylase may have evolved toward a larger Km(C02) and kcat than the C3 plant enzyme. Ribulose-P2 carboxylase from C4 plants does exhibit a rela- tively large Km (C02). I have attempted to correlate K (C02), k and the activation energy of the carboxylase m cat from several sources. A technical problem which was anticipated in this study was that purified ribulose-P2 carboxylase exhibits various specific activities from different organisms. The purified carboxylase from tobacco has been reported with specific activities varying between 0.1 to 0.5 (Brown et al., 1980; Kawashima et al., 1971; Kung et al., 1980; Koivuniemi et 110 al., 1980). The enzyme from other sources has likewise shown various specific activities (Bird et al., 1982). Few sources, such as spinach, are available which afford the. purification of ribulose-P2 carboxylase with specific activities of 2.0 to 2.3. Frequently inactivation due to protease digestion of carboxylase during purification has been proposed as an explanation of the poor specific acti- vity of the enzyme (Gatenby and Cocking, 1978; Harris and Stern, 1977; Peoples and Dalling, 1978; Sugiyama et al., 1968b), and recently investigators have taken care to include protease inhibitors such as leupeptin in their en-4 zyme extraction. To eliminate the problem of variable specific activity dependent on enzyme preparation, enzyme active site concen- tration was assessed by the ability to bind the transition state analogue, CABP (Hall et al., 1981; Pierce et al., 1982). The ability to bind CABP correlates with ribulose-P2 carboxylase activity when enzyme is denatured with heat (Hall et al., 1981). Unfortunately this procedure indicated widely different specific activities for various enzyme preparations. The effect of proteolysis on ribu- lose-P2 carboxylase activity and CABP binding was con- sequently examined. CABP binds with high affinity to ribu- lose-P2 carboxylase from spinach leaves after nearly total inactivation by proteolysis with trypsin. Therefore CABP binding may not be used as a measure of the catalytically 111 active sites where enzyme inactivation by protease digestion may be a factor. MATERIALS AND METHODS Ribulose-P2 carboxylase/oxygenase was purified from spinach leaves from a local market by ammonium sulfate frac- tionation, molecular sieve chromatography and ion exchange chromatography, as described elsewhere (McCurry et al., 1982). The enzyme from Egg gays c.v. Pioneer 3780, Glycine max or Chlamydomonas reinhardtii was purified as described ‘for the enzyme from spinach except for tissue homogeniza- tion. Extraction medium was as described for the prepara- tion of spinach ribulose-P2 carboxylase, except 0.4 mM dithiodiethyl-carbamate and 10 M leupeptin were included. Leaves from young maize plants (three weeks old) were cut into 1 cm squares and homogenized in an automated mortar and pestle (Torsion Balance Co, Clifton, NJ). Leaves from soy- bean were homogenized in a Waring blendor. Chlamydomonas cells were grown on 5% C02, collected by centrifugation and sonicated in a rosette cell. As judged by SDS-gel electro- phoresis, the proteins were 95% pure (in the case of the maize enzyme) to essentially homogeneous for the enzyme from other sources. Trypsin (292 units/mg protein) was obtained from Worthington and had been treated with N-p-toluenesul- fonyl-L-phenylalanine chloromethyl ketone to inactivate 112 chymotrypsin. This enzyme was dissolved at 0.5 to 5 mg/ml in 1 mM H2504. Trypsin inhibitor, type II-O from Sigma, was partially purified from chicken egg white. Ribulose-P2 was synthesized and purified as described in Chapter 1. CABP and 2'-14C-CABP were prepared as de- scribed elsewhere (Pierce et al., 1980). Other chemicals were of analytical reagent quality and used without further purification. Ribulose-P2 carboxylase/oxygenase was stored, activated and assayed as described in Chapter 1. Carboxylase assays (30 s) were initiated with the addition of activated enzyme (4 to 60 g) to a vial containing 0.25 ml with 0.1 M Bicine-Na+ (pH 8.2), 20 mM Mgc12, 10 mM NaHCO 1 mM DTT, 3. 0.5 mM ribulose-P2 and 0.2 mM EDTA. Trypsin inhibitor was included in all assays at 150 times (w/w) the amount of trypsin included. Kinetic determinations required varying the concentration of NaHCO3 (0.5 - 20 mM), the addition of carbonic anhydrase (0.010 mg/ml), maintenance of anaerobic conditions by gassing with N2 and hermetically sealing the vial, and reduction of assay duration to 15 or 20 s. Carboxylase assays were terminated with the addition of 0.1 ml of 2 N HCl. The vials were dried at 95 C and analyzed for incorporation of acid stable 14 C by scintillation count- ing. Ribulose-P2 oxygenase activity was assayed by the addi- tion of lOO‘pg of activated enzyme into the chamber of an 113 oxygen electrode containing 1 ml with 0.1 M Bicine-Na+, 20 mM MgC12, and 0.5 mM ribulose-P2. CABP binding to ribulose-P2 carboxylase was determined by a method described elsewhere (Pierce et al., 1982). Ribulose-P2 carboxylase/oxygenase (1 mg, 14.5 nmol active sites) was added to a solution containing 29 nmol l4C-CABP (0.5 Ci/mol), trypsin inhibitor at 150 times 2v- (w/w) the amount of trypsin included, 20 mM MgCl2 and 10 mM NaHCO3. After 45 minutes at 22 C, 290 nmol of unlabelled CABP was added, and the enzyme was incubated for at least 6 hours at 22 C. The protein was precipitated with 20% (w/v) polyethylene glycol 4000 and 20 mM MgCl2 and washed twice by resuspension of the protein in a solution of 20% polyethy- lene glycol and 20 mM MgC12. The protein was dissolved in '50 mM Bicine-Na+ (pH 8.2) and 5 mM NaHCO3 and counted 14C. for SDS-polyacrylamide gels were prepared with 15% acrylamide and 0.4% bis-acrylamide and electrophoresed with an LKB slab gel electrophoresis apparatus as described by Laemmli (1970). Gels were stained with 0.1% Coomassie Brilliant Blue R-250 in 50% ethanol and 10% acetic acid and destained with 17.7% (v/v) ethanol and 10% (v/v) acetic acid. Peptides and protein were assayed by reaction with fluorescamine (Udenfriend et al., 1972). Sample was diluted to 1 ml with H20 and 0.5 m1 of 0.2 M borate-Na+ buffer (pH 114 9.0) was added. Fluorescamine (0.5 ml, 0.2 mg/ml in ace- tone) was added with rapid mixing. The relative fluoreScence was assessed on an Aminco fluoro-colorimeter with the appropriate filters (390 nm excitation, 475 nm emission). RESULTS The kinetic parameters obtained with ribulose-P2 car- boxylase from several sources are presented in Table 3. The enzyme from plants that have mechanisms for the accumulation of CO2 (maize and Chlamydomonas) has a high Km (C02) com- pared to the enzyme from C3.p1ants (spinach and soybean). The values reported here reflect the true Km(C02), since the determinations were performed under anaerobic conditions. The Vmax value was calculated using a protein concentration based on the amount of CABP bound by a given volume of enzyme solution. Therefore, the protein concentration re- flects only the fraction of the protein which tightly binds CABP. Using this technique, approximately 4-fold variation was observed in specific activity of ribulose-P2 carboxylase between these organisms. There was no statistical differ- ence in the enthalpy of activation among these proteins. 115 Table 3.Summary of Kinetic Parameters of Ribulose-P2 Car- boxylase from Various Sources. *Km(C02) *Vmax(“m°1 AHf(kcal/mol) (pM) min'l mg'1 protein) spinach 30.5 1 2.2 2.87 1 0.08 12.7 1 0.87 soybean 19.6 1 1.3 0.78 1 0.05 12.1 1 3.10 Maize 44.4 1 6.3 1.49 1 0.08 12.7 1 0.49 Chlamydomonas 46.3 1 3.3 3.23 1 0.16 13.5 1 1.54 * At 30 C, note K (CO ) has similar temperature dependence as kcat; naturaT lo Km[C02] = 10,800 (l/T) + const. for the spinach enzyme. AH*was calculated from the slope of natural log [k /T] versus l/T over the range between 15 C and 35 C withakinetic analysis performed at 5 C inter- vals. Kinetic parameters were estimated with the aid of a computer program based on the method of Wilkinson (1969). The validity of correlating active enzyme with CABP binding was examined by testing the effect of proteolysis, since this might reflect a process which occurs during pro- tein purification. Digestion of activated ribulose-P2 car- boxylase/oxygenase with 0.5% (w/w) trypsin results in rapid loss of carboxylase activity (Fig. 18A). Ribulose-P2 oxy- genase activity declined similarly, and neither carboxylase or oxygenase activities were selectively inhibited (data not shown). The ability of the protein to bind the transition state analogue, CABP, was relatively insensitive to proteo- lysis by trypsin. After limited trypsin digestion, ' ribulose-P2 carboxylase still bound 70 to 80% of the CABP 116 Figure 18. The Effect of Trypsin an Activity and CABP Binding by Ribulose-P2 Carboxylase. A. Activated ribulose-P2 carboxylase (2 mg/ml) was treated with trypsin (0.01 mg/ml) at 30 C and assayed for carboxylase activity and the ability to bind CABP as proteolysis proceeded. The activity and ability to bind CABP recorded at time zero was assessed before the addition of trypsin. The theoretical activity was calculated by normal- izing the observed (actual) activity by the frac- tional binding of CABP, as calculated by Hall et a1. (1981). B. The data presented in Figure 18A is plotted with CABP binding as a function of catalytic activity for comparison with Figure 3 of Hall et al. (1981). ll7 .o ;p_3 umuo_uwcw mam; mcovpoomm .zuaz Co cowaccucmocoo 888.65 ea... .25 z: a: .35 :3 3232a 538... z: om 853:8 22:2 8:83. .ee: Locbcwocou mu_:mxu mo :o_uo::m o mm copr53m=ou No ucmucmamo aimcoumomxxocuxswo we mpom Po_u_:H .mm .mwd 136 (5 <5 0 c5 6 o o . 0 '0 <1- r0 8 9 (UIw/IowU) uoudwnsuoo 2o. lo 8103 IDIIIUI é NaCN (mM) Figure 23. 137 Fig. 24. Effect of pH on the Initial Rate of 02 Consumption with Ribulose-Pz and Cyanide. The reaction mixture was composed of 50 "M sodium phosphate, 50 mM sodium pyr0ph05phate, 0.6 mM ribulose—P2 and 0.5 mM NaCN. Reactions were initiated with the addition of cyanide. l38 m m asses cozaEBcoo No .6 90m .23 O alc. ISO ’ 7.0 Figure 24. 139 .m msmgom I :I m I one _._I -I...on_o oI I -deOIwII -Iwwo/ Loin /\oI II Inc +2.. :IIlmo (IwIOI III A: \lI menu/J zmolwll zmol IH_TI zI mIomlo_/I IoIM_TI Io I0 I 140 ferricytochrome c or NBTZT. Since the rate of cytochrome c or NBT2+ reduction was inhibited by MnSOD, the presence of 02- was indicated. The reaction of ribulose-P2 and cyanide with 02 resulted in the reduction of ferricytochrome c or NBTZT. Also, the addition of cytochrome c or NBTZT, which are reduced by 02- to regenerate 02, inhibited the rate of 02 consumption. These observations (data not shown) confirmed the results of Robertson et al., and suggested that 02- was involved in the mechanism of the reaction. The role of 02- in this reaction mechanism was corraborated by a study of the initial rate of 02 consumption during the oxidation of ribulose-P2 with cyanide in the presence of MnSOD. MnSOD was chosen for this study since it is not inhibited by cyanide, unlike the commercially available copper-zinc superoxide dismutase. The MnSOD preparation was void of catalase activity. Addition of increasing activity of MnSOD resulted in 49% inhibition of the initial rate of 02 consumption with cyanide and ribulose-P2 (Fig. 25) or dihydroxyacetone-P (data not shown). These results indicate that the initial product of 02 consumption was 02-, which could be enzymatically dismutated to H202 and 02. Since the Spontaneous rate of dismutation of 02- to H202 and 02 is appreciable at pH 8.5 (k2=104 M'1SEC'1) (Fee and Valentine, 1977), the 02- produced as a consequence of the oxidation of sugar-P in the presence of cyanide was unstable. One product of the dismutation, H202, was established in the reaction mixture by enzymatic analysis with catalase or peroxidase. Addition of catalase to the reaction, after about 90% of the 02 consumption had ensued, 141 .Ima we; come: to o_e= H Le m~.o se ee_owe_eez .oomez Le sb_>cooe eeoeo_eea ego eee zoez 2e H .Na-emepsa_c IE ~.H .Am.m Iav +ez-ee_o_m :2 om eee_ebeeo eesox_e :o_bomoc one .ameu25m_o mu_xocmazm mmmcmmcmz an cowuqezmcou No we mama _o_u_:H asp Co cowu_a_;cH .mm .m_d l42 L (quI/IouIU) uoIIduInsuog 9. 2o 10 emu IDIIIUI IO 02 CD '0 Q) .0 "C Qzo as» Ease uw>osmc mew: mucoacoo as» .msspxwa :o_uoomc m_;u mo coxop mm: :_mm voH x a ;p_2 :oow :_E a < .zuoz :5 H can .I Q; 0‘ 0' (Eq. 4) When DMPO was included in the ribulose-P2 and cyanide reaction mixture, only a trace of a signal (AN = 14.9 G, AHB = 14.8 G) could be observed (Fig. 27). However, if the reagents were not treated with a chelating resin or if 1 mM FeCl3 was included, the ribulose-P2 and cyanide reaction mixture yielded a strong signal (AN = 14.9 G, AHB = 14.8 G). When the reaction mixture contained ribulose-P2, the intensity of this signal was enhanced by about 50 fold by the addition of 1 mM FeCl3. With dihydroxyacetone-P, the signal enhancement was even greater (Fig. 27). The hyperfine splitting constants indicated that the hydroxyl radical (-0H) adduct of DMPO had been formed. An identical EPR spectrum was obtained when hydroxyl radical was generated by the Fenton reaction (Eq. 6) (Fenton, 1894). The presence of the hydroxyl radical adduct of DMPO may arise through the rearrangement of the superoxide adduct of DMPO or from the reaction of DMPO with hydroxyl radical (Finkelstein et al., 1979). In order to differentiate between these possibilities, the experiment was repeated in the presence of 0.1 M ethanol. Hydroxyl radicals will react with ethanol to produce o-hydroxyethyl radicals (Finkelstein et al., 1980). The inclusion of ethanol decreased the intensity of the hydroxyl radical spectrum, and a new signal was observed with the characteristic hyperfine Splitting constants of the o-hydroxyethyl adduct of DMPO (AN = 14.8 G, AHB = 22.8 G). Therefore the hydroxyl radical adduct of DMPO was derived from hydroxyl radical in 149 solution, and was not an artifact of rearrangement of some other DMPO adduct. The origin of the hydroxyl radical can be explained by the Haber-Weiss reactions (Haber and Weiss, 1934). Fe+3 + 02- —> Fe+2 + 02 (Eq. 5) Fe+2 + H202 _) Fe+3 + 'OH + -0H (Eq. 6) A similar mechanism for the production of hydroxyl radical from superoxide has been proposed by other investigators (Weinstein and Bielski, 1979; Halliwell, 1978; Buettner et al., 1978b; Buettner and Oberley, 1978a). Thus, the trapping of hydroxyl radical in the presence of Fe+3 implicates that both 02- and H202 had been formed in the oxidation of ribulose-P2 and cyanide by 02. No 02- adduct of DMPO was observed with the sugar-P and cyanide reaction mixture. Superoxide is not very reactive with DMPO and the superoxide adduct of DMPO is unstable under alkaline conditions (half-life = 353 at pH 8.0) (Finkelstein et al., 1980; Buettner and Oberley, 1978a). The ability to observe the 02- adduct of DMPO in the sugar-P and cyanide reaction mixture was checked by including NADH and riboflavin, which generated 02-. No 02- adduct of DMPO could be observed at pH 8.5. The 02- adduct of DMPO was observed at pH 7.6 and was dependent on NADH and riboflavin. These results appeared convincing that ribulose-P2 oxidation did . occur in this system, however, one outstanding observation could not be explained. Why was the 02 consumed only a fraction of the sugar-P included in the reaction? Robertson et'al. (1981) claimed that the 150 oxidation of the carbohydrate did go to completion as measured by cytochrome c reduction. To test the dependence of 02 consumption on ribulose-P2, ribulose-P2 was treated with activated ribulose-P2 carboxylase under catalytic conditions or a control solution which lacked enzyme. Carbon fixation was determined, and 100% of the ribulose-P2 was converted to product in the presence of enzyme. The addition of cyanide to either solution still gave identical rates and stoichio- metries of 02 consumption. Neither glycerate-3-P and glycolate-Z-P, products of the carboxylase and oxygenase reactions, exhibited cyanide dependent 02 consumption. Ribulose-Pz was treated with alkali to eliminate phosphate and to generate a diketo monophosphate product (Paech et al., 1978) and this preparation was no more reactive with cyanide in 02 consumption then ribulose-P2. Therefore, 02 consumption dependent on the addition of cyanide to ribulose-P2 solutions was not due to ribulose-P2, but was presumably due to a contaminant present in the ribulose-P2 preparation. Discussion The substrate present in ribulose-P2 and other solutions of sugar-P which reacts with cyanide and 02 is unknown. The results show that 02 may be reduced by one electron to 02'. This was indicated by the inhibition of 02 reduction by MnSOD, NBTZ+ and cytochrome c. Hydrogen peroxide was also a product of the reaction, presumably via the dismutation of 02‘, as indicated by the peroxidase coupled reaction and the release of 02 after the addition 151 of catalase to the reaction mixture. The addition of ferric iron to .the reaction generated hydroxyl radical, as predicted by the Haber-Weiss reactions if both 02' and H202 were present. Although the nature of the substrate for this reaction is unknown, the addition of cyanide generates a reductant capable of reducing 02 to 02'. The redox potential for the reduction of 02 to 02' is approximately -0.3 V (Fee and Valentine, 1977). The contaminant was present in each of several ribulose-P2 preparations. Two preparations of dihydroxyacetone-P from Sigma also showed reactivity with 02 in the presence of cyanide. Cyanide reacts with carbonyls to form nitriles. The cyano substituent is strongly electron-withdrawing and the pK of an a proton will be decreased. The increased acidity of the cyano adduct may generate carbanions, which in principle could react.as proposed by Robertson et al. (1981) in Scheme 5. Although this scenario represents a potential mechanism, the explanation for the non-stoichiometric reaction with 02 remains obscure. CHAPTER 7 THE EFFECT OF SOME POTENTIALLY REGULATORY COMPOUNDS ON RIBULOSE-P2 CARBOXYLASE/OXYGENASE. Ribulose-P2 carboxylase/oxygenase catalyzes the first committed reaction for each of two important metabolic path- ways, photosynthesis and photorespiration. The regulation of the carboxylase and oxygenase activities and the ratio of these activities are important parameters which dictate photosynthetic performance. As summarized in the literature review, the only known factors effecting the ratio of car- boxylase to oxygenase are the tensions of CO2 and 02' temperature, and the metal ion used in activation. Only the two former parameters are physiologically important. Never- theless, any compound or condition which might regulate the activities or the ratio of ribulose-P2 carboxylase/oxygenase would be of mechanistic interest to basic scientists and agronomic interest to applied scientists. This chapter summarizes the effect of several compounds on ribulose-P2 carboxylase/oxygenase which have potential as regulators of the enzyme activity. Materials and Methods Ribulose-P2 carboxylase/oxygenase was purified, stored, activated and assayed as described in Chapter 1. Ribulose-P2 was synthesized and purified as described in Chapter 1. 152 153 Fructose 2,6-P2 was a gift from Dr. Simon J. Pilkis, Department of Physiology at the Vanderbilt University, Nash- ville, Tennesee. Trans-Z-hexenal was obtained from Aldrich. Ribose-S-P was obtained from Sigma. Results and Discussion The Effect 91 Fructose-2,6-Pa on Ribulose-ngCarboxylase/Oxygenase. Fructose-2,6-P2 is a potent activator of ATP-dependent phosphofructokinase from several sources (Hers and Van Schaftingen, 1982), a potent activator of the PPi dependent phosphofructokinase from mung beans (Sabularse and Anderson, 1981), and a potent inhibitor of fructose bisphosphatase from several sources (Hers and Van Schaftingen, 1982). The possibility that this compound also exerts some regulatory effect on ribulose-P2 carboxyl- ase was tested. Activated ribulose-P2 carboxylase was weakly inhibited by fructose-2,6-P2 under the standard assay conditions (Table 4). Inhibition was greater when ribulose-P2 was de- creased to twice Km (0.05 mM). This response suggests that fructose-2,6-P2 was a competitive inhibitor with respect to ribulose-P2, but a more thorough kinetic analysis was not performed. Ribulose-P2 oxygenase was apparently not effect- ed by fructose-2,6-P2, but the standard deviations were high in these measurements. 154 The effect of fructose-2,6-P2 was also tested on acti- vation of ribulose-P2 carboxylase (Fig. 28). Effectors of the enzyme activation state are typically weak competitive inhibitors with respect to ribulose-P2 and modify the acti- vation state only under suboptimal activation conditions (eg. 1 mM HC03-) (McCurry et al., 1981). Fructose-2,6-P2 behaved in a manner typical of an effector with 39% stimulation of carboxylase activity observed at 0.3 mM fructose-2,6-P2. This response is weaker than that ob- served with gluconate-6-P which resulted in 110% stimulation of carboxylase activity at 0.5 mM. Fructose-2,6-P2 appears not to have any unusual effects on ribulose-P2 car- boxylase/oxygenase and is probably not involved in the regu- lation of this enzyme i vivo. Table 4. The Effect of Fructose-2,6-P2 on Ribulose-P2 Carboxylase/Oxygenase. [Ribulose-PZ] [Fructose- Rate (gmol'min 1' % of 2,6-P2] mg 1protein) control FM AM A.Carboxylase 500 0 1.87 1 0.08 100 500 100 1.67 1 0.05 89 50 0 1.13 1 0.06 100 50 100 ' 0.65 1 0.05 45 B.Oxygenase 500 0 0.112 1 0.008 100 1 0.012 100 500 100 0.112 l55 Figure 28. The Effect of Fructose-2,6-P2 on Ribulose-P2 Carboxylase Activation. Ribulose-P2 carboxylase was incubated under stand- ard activation conditions except that NaHCO3 was 1.0 mM and fructose-2,6-P2 was included. Aliquots of enzyme (40 pg) were assayed under standard conditions (10 mM NaHCO3) for 15 s to minimize activation during the assay. mg”1 protein 1 pmol 002 -min l56 l 1.45 l 1.30 1.15 I 1.00 0.85 0.0 T 0.1 012 0:3 UT4 [Fructose-2,6-P2] mM Figure 28. 0.5 157 The Effect 91 Trans-Z-hexenal 93 Ribulose-P31Carboxylase/Oxygenase. Trans-Z-hexenal was tested on ribulose-P2 carboxylase/oxygenase for two reasons. First trans-Z-hexenal is present in millimolar concentra- tions in crude plant extracts and is responsible for the distinctive odor of freshly cut grass. This could represent a physiologically important compound. However further literature search on this topic indicated that it was not actually present in plants but rather was generated as a metabolite in crude leaf extracts due to lipoxygenase acti- vity (Spoehr and Nye, 1943). Secondly, an abstract at the 1982 Federation of American Societies for Experimental Biol- ogy (Martin and Tabita, 1982) reported that activated enzyme 'incubated in the presence of aldehydes showed an increased carboxylase to oxygenase ratio. Glyoxylate has been demon- strated not to selectively inhibit carboxylase or oxygenase activity (Chapter 3). Trans-Z-hexenal was added to acti- vated enzyme at 1.25 mM and activity was followed over one hour (Fig. 29). The inhibition followed psuedo-first order kinetics with a rate constant for inhibition of 0.021 min-1 for each activity. Therefore trans-Z-hexenal demon- strated no selectivity for inhibition of ribulose-P2 car- boxylase or oxygenase. No physiological role for trans-Z-hexenal may be postulated, since this compound is produced in crude plant extracts and is not present in 158 Figure 29. The Effect of Trans-Z-hexenal on Ribulose-P2 Carboxylase and Oxygenase Activities. Trans-Z-hexenal (100 mM) was prepared in absolute ethanol and added to enzyme at 1.25 mM. The final concentration of ethanol was 0.8% (v/v). Aliquots were taken and assayed for carboxylase and oxygen- ase activities by the standard assays. Carboxylase assays were performed at each time point in duplicate. Oxygenase assays are single determinations. |n(% activity) 3.00 3.25 3.50 3.75 O l 4c00 4025 4050 l l l l l l59 I T T 20 30 40 time (min) Figure 29. 160 plants per se. The Effect 91 Ribose-S-P 93 Ribulose-P2 Carboxylase/Oxygenase. Ribose-S-P is a negative effector of ribulose-P2 carboxylase/oxygenase (McCurry et al., 1981). Apparently it binds more tightly to the un- activated form of the enzyme and draws the equilibrium away from activation. Glycolate-Z-P phosphatase is also inhibited by ribose-S-P in a manner indicative of partial competitive inhibition with respect to glycolate-Z-P (H. David Husic, personal communication). A plot of appar- ent Km (glycolate—Z-P) versus ribose-S-P concentration showed a nonlinear trend at high inhibitor concentration. The inhibition of ribulose-P2 carboxylase/oxygenase by ribose-S-P was examined (Fig. 30). The inhibition by ribose-S-P was essentially competitive with a KI of 5.4 mM. Some deviation from competitive inhibition is noted at the highest concentration of inhibitor (10 mM); Vmax was dimin- ished by 18% under these conditions. This may be the result of ribose-S-P acting as a negative effector and decreasing the activation state of the enzyme and consequently Vmax' The plot of apparent Km (ribulose-P2) versus inhibitor is relatively linear over the range of inhibitor concentration tested (Fig. 30). The response of ribulose-P2 carboxylase to ribose-S-P may be explained as a combination of competitive inhibition and deactivation due to the negative effector phenomenon. 161 Figure 30. The Inhibition of Ribulose-P2 Carboxylase by Ribose-S-P. Enzyme (4 pg) was activated and assayed under standard conditions with variable ribulose-P2 con- centration (58-690 pM) and variable ribose-S-P concentration (0 to 10 mM). Assays were performed in triplicate and kinetic parameters were analyzed by the method of Wilkinson (1969) with the aid of a computer program. These data indicate a K of I 5.4 mM for ribose-S-P. l62 m.a E395P 0:... SE. .081 .be> xi I 4 ll PII N.“ 0.0 v.0 0.0 p . _ _ 0 -m 5 7m In. h_r In... 5 I O "m PI [ rem 2 K 0 _ b a lid nw ow ow 0v ON 0 21 .A malomoSntvex Embaaaa 0.. I .0 Figure 30. 163 Ribose-S-P is a poor inhibitor of ribulose-P2 carboxylase and probably plays no physiological role for regulating this enzyme. List 91 Publications Cook, C.M., R.M. Mulligan and N.E. Tolbert. The Stimulation and Inhibition of Spinach Ribulose Bisphosphate Carboxylase/Oxygenase by Glyoxylate. Manuscript in Preparation. Mulligan, R.M. and N.E. Tolbert. The lability of an Intermediate from the Ribulose Bisphosphate Carboxylase Reaction. Manuscript accepted by Archives of Biochemistry and Biophysics. Mulligan, R.M., B. Wilson and N.E. Tolbert (1983). The Effects of Glyoxylate on Photosynthesis by Intact Chloroplasts. Plant Physiology (In Press) Pierce, J.W., S.D. McCurry, R.M. Mulligan and N.E. Tolbert (1982). Activation and Assay of Ribulose Bisphosphate Carboxylase/Oxygenase. Methods in Enzymology 82:47-55. Mulligan, R.M. and N.E. Talbert (1980). Properties of a Membrane-Bound Phosphatase from the Thylakoids of Spinach Chloroplasts. Plant Physiology §§:1169-1173. Mitchell, M.J., R.C. Hartenstein, B.L. Swift, E.F. Newhauser, B. Abrams, R.M. Mulligan, B.A. Brown, D. Kaplan (1978). Effects of Different Sewage Sludges on some Chemical and Biological Characteristics of Soil. Journal of Environmental Quality 1:551-559. Hartenstein, R., E.F. Neuhauser and R.M. Mulligan (1977). Mechanism of Action of Lignin and Lignin-Model Compounds with Horseradish Peroxidase. Phytochemistry 1§:l855-1857. Mitchell, M.J., R.M. Mulligan, R. Hartenstein, and E.F. Neuhauser (1977). Conversion of Sludges into "Topsoils" by Earthworms. Compost Science 18:28-32. Abstracts a1_Meetings Mulligan, R.M. and N.E. Tolbert (1981). Enzymatic Mechanism of Ribulose Bisphosphate Carboxylase/Oxygenase. Plant Physiology 675:189.. Gee, R., R.M. Mulligan and N.E. Tolbert (1981). 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