Kif‘éETiC, BINDING, ANS CDPEFGRMATEONAL STUDES OF YEAST PYRWATE KINASE Thesis for the Degree of Ph; D. MECHEGAR STATE UEinVERSETY lAMES RAY HUNSLEY 1970 LIB RA R Y Michigan Sta to University J This is to certify that the thesis entitled KINETIC, BINDING, AND CONFORMATIONAL STUDIES OF YEAST PYRUVATE KINASE presented by James Ray Hunsley has been accepted towards fulfillment of the requirements for _P_h..lL__degree tummy //m_ M Major professor Date February 16, 1970 0-169 ABSTRACT KINETIC, BINDING, AND CONFORMATIONAL STUDIES OF YEAST PYRUVATE KINASE BY James Ray Hunsley A homogeneous preparation of bakers' yeast pyruvate kinase (EC 2.7.1.40) has been shown to exhibit coOperative steady—state kinetics for the stringently required mono- valent cations K+ and NH4+, Mgz+ , and phosphoenolpyruvate, but not for ADP, at saturating concentrations of all other substrates and metal ions, pH 6.2. Fructose-1,6-diph03phate was shown to heterotropically activate the enzyme, trans- forming the sigmoid saturation curves for monovalent cation, 2+ Mg , and phOSphoenolpyruvate to hyperbolic without affect— ing the Vm. In the presence of fructose diphosphate, Na+ 2+ 2+ and Ca were able to replace K+ or NH4+ and Mgz+ or Mn but at less efficiency. Inclusion of fructose diphosphate as well as increasing the phosphoenolpyruvate concentration increased activity of the enzyme in the basic pH range. Strong kinetic interactions were noted for phosphoenol diphothate centrations quired to D The Mr nary comple rate and ei atypical b: 0f fructosl action, cf on the bin nary compl filled Mn2 James Ray Hunsley phosphoenolpyruvate or ADP and an+ at pH 7.5. Fructose diphosphate abolished all ADP—Mn interactions, but high con— centrations of phosphoenolpyruvate were additionally re— quired to uncouple phosphoenolpyruvate-Mn interactions. The Mn-enzyme and Mn-enzyme-substrate binary and ter— nary complexes were studied by the NMR proton relaxation rate and electron paramagnetic resonance techniques. An atypical binding curve for Mn2+ in the presence or absence of fructose diphosphate was suggestive of site-site inter- action. Comparison of the effects of fructose diphosphate on the binding parameters of the phosphoenolpyruvate ter- nary complexes was consistent with the requirement of one filled Mn2+ binding site before the effector could lower the affinity for the substrate. As implied from comparison of binding and kinetic data, the enzyme—metal complex may bind phosphoenolpyruvate prior to the binding of ADP. Preparative isoelectric focusing and microisoelectric focusing in polyacrylamide gel columns have resolved two conformers of the enzyme. Fructose diphosphate was shown to Specifically convert the conformer of low isoelectric point to the high pH form which, upon removal of the effec- tor, reverted to a mixture of both conformers. No kinetic differences were demonstrable for the two conformers and the tetrameric tained. Metal | moles of t g of prote enzyme has the rabbit acid conte James Ray Hunsley tetrameric structure of the native enzyme was probably re- tained. Metal analyses have revealed the presence of 0.14 moles of tightly bound Cu2+ of unknown function per 166,000 g of protein. Amino acid analysis has shown that the yeast enzyme has a relatively low aromatic amino acid content like the rabbit muscle enzyme but deviates significantly in amino acid content. KINETIC, BINDING, AND CONFORMATIONAL STUDIES OF YEAST PYRUVATE KINASE BY James Ray Hunsley A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1970 66.2776: 7r/h,7o ACKNOWLEDGEMENT I thank Dr. Clarence H. Suelter who provided the freedom and support required to make this thesis a reality. Help from Miss Betty Baltzer, Miss Doris Bauer, Mrs. Kathy Ashworth, Mr. Bob Brown, Mr. Ron Kuczenski, Dr. John LaRue, Dr. John Wilson, Dr. Albert Mildvan, and Dr. Thomas Vogel at various stages of the work is gratefully appreci— ated. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . 1 LITERATUREREVIEW................ 3 I. Reaction mechanism and kinetic control . 4 1. Random versus ordered binding . . . 4 2. Metal complexes and the nature of the true substrates . . . . . . . . 5 3. Activators . . . . . . . . . . . . . 6 4. Inhibitors . . . . . . . . . . . . . 10 5. Kinetic cooPerativity . . . . . . . 12 6. Temperature effects . . . . . . . . 15 II. Molecular heterogeneity and regulation of enzyme levels . . . . . . . . . . . . 15 1. Nutritional and hormonal control . . 15 2. Isozymes and multiple forms . . . . 17 3. Conformers and introconvertibility of forms . . . . . . . . . . . . . . 20 METHODS AND MATERIALS . . . . . . . . . . . . . . 23 I. Enzymes . . . . . . . . . . . . . . . . . 23 II. Chemicals . . . . . . . . . . . . . . . . 25 III. Experimental methods . . . . . . . . . . 26 1. Assay of activity and determination of enzyme concentration . . . . . . 26 2. Electrophoresis and chromatography . 27 3. Chemical prOperties . . . . . . . . 28 4. Kinetic properties . . . . . . . . . 29 5. Binding properties . . . . . . . . . 30 6. Isoelectric focusing . . . . . . . . 31 RESULTS . . . . . . . . . . . . . . . . . . . . . 34 I. Criteria for purity . . . . . . . . . . . 34 1. Disc gel electrophoresis . . . . . . 34 2. Contaminating enzyme activities . . 37 iii iv II. Chemical prOperties . . . . . . . . . . 1. Extinction coefficient . . . . . . 2. Amino acid analysis . . . . . . . 3. Metal content . . . . . . . . . . 4. Gel chromatography . . . . . . . . III. Kinetic properties . . . . . . . . . . 1. Requirement of monovalent cation . 2. Kinetics of the Mg2+ activated system . . . . . 2+. . . . . . . . 3. Kinetics of the Mn activated system . . . . . . . . . . . . . . 4. Na—FDP kinetic interactions . . . 5. Mn-substrate interactions . . . . 6. Miscellaneous kinetic observations IV. Binding properties . . . . . . . . . . l. Proton relaxation rate and electron paramagnetic resonance studies . . 2. Lack of tight FDP binding . . . . V. Isoelectric focusing . . . . . . . . . 1. Preparative focusing . . . . . . . 2. Microisoelectric focusing . . . . DISCUSSION 0 O O O O O O O O O O O O O O O O O 0 SUMMARY 0 O O O O O O O O O C O O O O O O O 0 LIST OF REFERENCES . . . . . . . . . . . . . . . Page 37 37 38 38 41 41 41 43 55 62 73 84 84 84 90 93 93 93 103 114 116 LIST OF FIGURES Figure 1. 10. Disc gel electrophoresis of 50 pg of yeast pyruvate kinase . . . . . . . . . . . . . . . Relationship between initial velocity of yeast pyruvate kinase and activating univalent cation concentration . . . . . . . . . . . . . Relationship between initial velocity of yeast pyruvate kinase and total FDP concentration . . . . . . . . . . . . . . . . Relationship between initial velocity of yeast pyruvate kinase and total PEP concentration . . . . . . . . . . . . . . . . Relationship between initial velocity of yeast pyruvate kinase and total MgClz concentration . . . . . . . . . . . . . . . . Relationship between initial velocity of yeast pyruvate kinase and total ADP concentration . . . . . . . . . . . . . . . . Relationship between initial velocity of yeast pyruvate kinase and KCl or NH4C1 concentrations . . . . . . . . . . . . . . . . The effect of pH and PEP concentration on the activity of yeast pyruvate kinase . . . . Relationship between initial velocity of yeast pyruvate kinase and total NaCl or KCl concentration . . . . . . . . . . . . . . . . Relationship between initial velocity of yeast pyruvate kinase and total FDP concentration . Page 37 45 48 50 52 54 57 59 61 64 vi Figure 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. Relationship between initial velocity of yeast pyruvate kinase and total PEP concentration . . . . . . . . . . . . . . . Relationship between initial velocity of yeast pyruvate kinase and total MnC12 concentration . . . . . . . . . . . . . . . Relationship between initial velocity of yeast pyruvate kinase and total MnC12 concentration . . . . . . . . . . . . . . . Relationship between initial velocity of yeast pyruvate kinase and total ADP concentration . . . . . . . . . .7. . . . . Lineweaver-Burk plot of effect of NaCl on Vm of yeast pyruvate kinase at infinite FDP concentration . . . . . . . . . . . . . Relationship between NaCl concentration and KA of yeast pyruvate kinase for FDP . . . . Relationship between Hill slope for Mn and total ADP concentration for yeast pyruvate kinase . . . . . . . . . . . . . . Relationship between total ADP concentration and apparent KA of yeast pyruvate kinase for MnCIZ o o o o o o o o o o o o o o o o 0 Relationship between Hill slope for ADP and total Mn concentration for yeast pyruvate kinase . . . . . . . . . . . . . . Relationship between total Mn concentration and apparent Km of yeast pyruvate kinase for ADP O O O O O O O O O O O O O O O O O 0 Relationship between Hill slope for Mn and total PEP concentration for yeast pyruvate kinase . . . . . . . . . . . . . . Page 66 68 7O 72 75 75 77 77 79 79 81 vii Figure Page 22. Relationship between total PEP concentration and apparent KA of yeast pyruvate kinase for Mnclz O O O O C O O O O O O O O O O O O O 81 23. Relationship between Hill slope for PEP and total Mn concentration for yeast pyruvate kinase C O O O O O O O O O O O O O O O O O O 83 24. Relationship between total Mn concentration and apparent Km of yeast pyruvate kinase for PEP O I O O O O O O O O O O O O C O O O O 83 25. Scatchard plot of the binding of manganese to yeast pyruvate kinase . . . . . . . . . . 86 26. Lack of binding of FDP to yeast pyruvate kinase . . . . . . . . . . . . . . . 92 27. Preparative isoelectric focusing of yeast pyruvate kinase . . . . . . . . . . . . 95 28. Microisoelectric focusing of yeast pyruvate , kinase in polyacrylamide gel columns . . . . 98 29. Microisoelectric focusing of yeast pyruvate kinase in polyacrylamide gel columns . . . . 100 Table l. Preper 1 Multi; presu: 3. Amino kinase 4. Metal pyruva 5- Requir for a1 kinase LIST OF TABLES Table Page 1. Pr0perties of cooperative pyruvate kinases . . 13 2. Multiple forms of pyruvate kinase and presumptive isozymes . . . . . . . . . . . . . 18 3. Amino acid analysis of yeast pyruvate kinase C O C O O O O O O O C O O O O O O O O O 39 4. Metal content of purified yeast pyruvate kinase C O O O O O O O O O O O O O O 40 5. Requirement of yeast pyruvate kinase activity for alkali metal or ammonium ions . . . . . . 42 6. Binary and ternary complexes of yeast pyruvate kinase with manganese and substrates . . . . . 88 viii ADP AMP ATP CHA EDTA EPR J :15 \? “ADE 53313 PPp P-VSp Tris ADP AMP ATP EDTA EPR FDP NADH NMR PEP PMSF Tris LIST OF ABBREVIATIONS AND SYMBOLS adenosine—5'-diphosphate adenosine-S'-monophosphate adenosine-S'-triphosphate cyclohexylammonium (cation) enhancement value enhancement value of binary Mn-enzyme complex ethylenediamine tetraacetic acid electron paramagnetic resonance fructose-1,6—diphosphate dissociation constant of metal—enzyme complex dissociation constant of ligand for metal—enzyme complex Hill slope nicotine adenine dinucleotide, reduced nuclear magnetic resonance phosphoenolpyruvic acid phenylmethanesulfonyl fluoride tris(hydroxymethyl)amino methane ix Tb phospbc action: from ra Studied ed to C reports trol p( twin 0; ulated and Ma (1964) Vided . Yeast repOrt INTRODUCTION The glycolytic enzyme pyruvate kinase (ATP pyruvate phosphotransferase, EC 2.7.1.40) catalyzes the following re— action: H+ + PEP + ADP :2 pyruvate + ATP. Preparations from rabbit muscle have, by far, been the most extensively studied though, in recent years, work has been widely extend— ed to other organisms. This interest has been stimulated by reports that have established the enzyme as a glycolytic con— trol point in several tissues and that have uncovered a spec— trum of mechanisms by which pyruvate kinase activity is reg- ulated. An earlier investigation of the yeast enzyme (Washio and Mano, 1960) was hampered by instability. Later, Hommes (1964), Pye and Eddy (1965), and Hess and Brand (1965b) pro- vided proof that the reaction was rate limiting in whole yeast or extracts. At the same time, the latter workers reported fructose-1,6-diphosphate activation of the enzyme. The function of this effector as a heterotropic activator toward the required monovalent and divalent cations and PEP was shown with purified enzyme by Hess gt 1. (1966), Hunsley and Suelter (1967), Hess and Haeckel (1967), Haeckel 1 2 ._£._l- (1968), and Hunsley and Suelter (1969b). Since convenient, stable preparations of high purity yeast pyruvate kinase in gram amounts are now available (Haeckel £5 31., 1968; Hunsley and Suelter, 1969a), it was chosen for study in an attempt to arrive at the origins of its cooperative effects and to compare the structural and functional relationships of it and the rabbit muscle enzyme which diSplays no allosteric behavior. Preliminary reports of this work have been presented (Hunsley and Suelter, 1967, 1969a, 1969b; Mildvan gt al., 1970). k1 aptly intern ccnver since, formed toward aCtivi Perple been a. and ti ( 301m 8 in int. LITERATURE REVIEW AsEects pf control of pyruvate kinase The enzyme pyruvate kinase lies at a critical point, aptly designated the "pyruvate crossroads" (S015, 1968), in intermediary metabolism because of the role it plays in the conversion of phOSphoenolpyruvate to pyruvate. In addition, since ATP, the chief primer of biosynthetic reactions, is formed in the reaction and the reaction is diSplaced far toward pyruvate (McQuate and Utter, 1959), the enzymatic activity might be expected to be carefully regulated. A perplexing number of possible controls have, in fact, now been documented for the enzyme from a variety of organisms and tissues. In both intact yeast cells and lysates (Hommes, 1964; Pye and Eddy, 1965; Hess and Brand, 1965b), in intact perfused rat heart under certain conditions (Williamson, 1965), and in guinea pig cerebral cortex slices (Takagaki, 1968), the pyruvate kinase reaction can be shown to be a glycolytic control point. Of equal importance are the reports of the probable modulation of the enzyme activi— ty during gluconeogenesis in yeasts and liver (Fernandez 35 .al., 1967; Sillero gt 1., 1969; Weber gt 31., 1967a). 3 4 This review will attempt to classify some of the literature on this subject which has appeared since the last exhaustive review of pyruvate kinase (Boyer, 1962). I. Reaction mechanism and kinetic control 1. Random versus ordered binding Mutually independent Michaelis constants from steady- state kinetic studies of substrates for an enzyme may be taken as presumptive evidence that binding events are ran- dom. Kinetic data for rabbit muscle (McQuate and Utter, 1959; Reynard £2 31., 1961), types M and L rat liver (Tanaka 33 11., 1967), and both forms of the crude rat epi- didymal fat pad (Pogson, 1968) pyruvate kinases are consis— tent with a random binding mechanism for ADP and PEP. Re— sults from the human erythrocyte enzyme are in conflict (Campos 35 31., 1965; Ibsen §£_§1., 1968) perhaps due to isozymic differences resulting from purification. A slight dependence of Hill slope and apparent Km of ADP on PEP con- centration was found by Haeckel_g§ a1. (1968) with a prep— eration from brewers' yeast (Saccharomyces carlsbergensis). Mildvan and Cohn (1966) have shown excellent agreement of binding and kinetic constants of the substrates for the rab— bit muscle enzyme substantiating a preferred pathway for ADP and PEP binding. Inconsistencies in kinetic and binding data for bakers’ yeast pyruvate kinase (Mildvan gt 31., 1970) may mean that this enzyme has a preferred order of binding PEP. 2. Metal complexes and the nature of the true substrates ADP and PEP readily form divalent and monovalent cation complexes (Melchior, 1954; Smith and Alberty, 1956; Wold and Ballou, 1957) within the physiological range of concen— +, and Na+. tration of the more important cations, Mgz+, K Furthermore, the secondary acid dissociation constants of the phosphate moities of ADP and PEP have pKa values of 6.7 and 6.4, reSpectively, and, at least for ADP, these values are dependent on the nature of the metal complex (WOld and Ballou, 1957: Smith and Alberty, 1956). Because of the lim- itations of equilibrium kinetics and the complexity of de— termining the various equilibria involving the substrates, studies of the possible catalytically active Species of sub- strates have been limited. For the rabbit muscle prepara— tion, Melchior (1965) concluded that MgADP‘ was the princi— pal interacting ADP species with the enzyme and that KADPZ' could be varied over a wide range with no effect on rate. In binding and kinetic studies with the same enzyme, Mildvan and Cohn (1966) saw no conflict with the hypothesis of ran- 2+ dom binding of Mn , ADP, and MnADP'. A computer simulation of the enzyme (Kerson 25 a1., 1967) was consistent with the 6 Mildvan and Cohn model, and indicated, because of the limi— 2+ tations of free Mg in cells, that activity was controlled by Mg2+ availability. In a reply to a criticism (Cleland, 1967) of this model, Mildvan gt_§1. (1970) in a rapid quenching kinetic approach to the problem, showed conclu— sively that the reaction, NiADP + enzyme, was not the only productive pathway to a quaternary complex. 3. Activators Monovalent cation—A pyruvate kinase from Acetobacter xylinum (Benziman, 1969) has now been described which shows no requirement for the monovalent cations K+ or NHJZ Data for two other bacterial sources are suggestive (Maeba and Sanwal, 1968; Ozaki and Shiio, 1969) but inconclusive be— cause of NHéland Na+ contamination. It would be of interest to know if both the constituitive and inducible pyruvate ki— nases of Escherichia coli require K+ for activity (Malcovati and Kornberg, 1969). The activation mechanism of monovalent cations is under controversy. The predominant thinking is that monovalent cations are necessary to maintain an enzyme conformation necessary for catalysis (Evans and Sorger, 1966). More re- cently, Suelter (1970) has theorized a specific mechanistic role for these cations: interaction with the enol-keto tau- tomers of pyruvate at the active site. Results exist which 7 support both hypotheses; a variety of phenomena such as en- zyme stability, sedimentation, electrophoretic behavior, fluorescence, and binding studies (Wilson 31 31., 1967; Sorger et 1., 1965; Mildvan and Cohn, 1964; Kayne and Suelter, 1965: Suelter 31 1., 1966; Suelter, 1967), have been interpreted via the conformational model. Recently, however, Kayne and Reuben (1970) have shown by direct nu— clear magnetic resonance that the activating univalent cat- ion 2osTh+1 binds rabbit muscle pyruvate kinase in very close proximity to the diamagnetic Mn2+ site, and therefore, the active site itself. Kinetically, strong interaction occurs between the allosteric activator FDP and K+ for yeast pyruvate kinase (Hess and Haeckel, 1967; Hunsley and Suelter, 1969b). NMR proton relaxation rate binding studies have shown that for the bakers' yeast enzyme, K+ raises the affinity of the enzyme-Mn complex for ADP (Mildvan 31 31., 1970). pH-Mechanistically, little can be said about the ef- fects of pH on pyruvate kinase activity. It should be noted, however, that a proton is taken up in the reaction in the direction of pyruvate formation. Rose (1960) concluded that the ATP—activated enolization of pyruvate catalyzed by the rabbit muscle enzyme, which rose steeply above pH 8, was dependent on the ionization state of the protein since no 8 new ionizing groups were available in the nucleotide above pH 7. With two cooperative enzymes from yeast and rat liv— er (Haeckel 33 31., 1968; Rozengurt 3; 31., 1969), varia— tions in the Km for PEP were pH dependent (although in an opposite manner) and pKa values of 7.0 and 6.9, respective— ly, could be extrapolated from the data for the ionizable groups reSponsible. These observations fail to explain, however, the double peaked pH activity profiles seen for several cooperative pyruvate kinases from brewers' and bakers' yeast, desert locust fat body, and rat liver (Haeckel 3; 31., 1968; Hunsley and Suelter, 1969b; Bailey and Walker, 1969; Bailey 31 _1., 1968). Metabolica11y derived activators-Fructose-l,6-diphos— phate was first identified by Hess and Brand (1965a) as an allosteric activator of yeast pyruvate kinase. The enzyme from many sources, including rat liver, loach embryos, E. 2211, fish muscle, human erythrocytes, mouse liver, fish embryos, Euglena gracilis, pig and chicken liver, and des- ert locust fat body and flight muscle (Tanaka 33 31., 1967; Milman and Yurowitzki, 1967; Maeba and Sanwal, 1968; Somero and Hochachka, 1968; Koler and Vanbellinghen, 1968; Carminatti 35 31., 1968; Mil'man and Yurovitskii, 1969; Ohmann, 1969; Leveille, 1969; Bailey and Walker, 1969) has similarly been shown to be FDP activated. 9 For most cases reported, FDP acts as a heterotrOpic allosteric activator by lowering the Hill constant for PEP and both required metal ions and lowering the apparent Km or Kg for each (Taylor and Bailey, 1967; Haeckel 31 31., 1968; Hunsley and Suelter, 1969b). There are at least two exceptions to this generalization and, for the first, a regulatory pyruvate kinase from E. 3311 (Maeba and Sanwal, 1968), FDP affected only the Vmax of the reaction but not the sigmoidality of the PEP rate concentration curve. AMP was reaponsible for the PEP Km change and converted PEP ki— netics from sigmoid to hyperbolic. For the second, a prep— aration from rainbow trout muscle (Somero and Hochachka, 1969), the PEP saturation curves were stated to have re— mained hyperbolic under all conditions. The Brevibacterium flavum enzyme (Ozaki and Shiio, 1969) is also AMP activated with no effect on PEP sigmoidicity. In fish embryos, a previously unreported positive effector, 3',5'-cyc1ic AMP, has been found (Milman and Yurowitzki, 1967; Mil'man and Yurovitskii, 1969), but no kinetic mechanism stated. The very small stimulation of yeast pyruvate kinase activity by ATP (Haeckel 33 31., 1968) was probably due either to FDP contamination or the complexing of a divalent metal inhib— itor. lO 4. Inhibitors Divalent metal ions—An elegant rationale for predic- 2+ inhibition of divalent metal ting the likelihood of Ca requiring enzymes which utilize nucleotides and other phos— phate substrates has been presented by Cohn (1963): en— zymes which do not require a metal atom to function as a bridge atom between enzyme and substrate, but only to bind 2+ the nucleotide, are Ca activatable. Enzymes with a req— uisite for direct metal binding are inhibited by Ca2+. Ternary complexes with a bridging divalent metal atom of the last class have been proven now for two pyruvate kin— ases, from rabbit muscle and bakers' yeast (Mildvan and Cohn, 1966; Mildvan 33,31., 1970). The mechanism of the Ca2+ inhibition is complex. 2+ Kachmar and Boyer (1953) reported Ca to give both com— petitive and non-competitive inhibition in relation to K+. For the muscle enzyme, proton relaxation measurements dem— 2+ 2+ onstrating that Ca strictly competed for Mn in the metal-enzyme complex significantly were made in the pres- ence of 0.10 M KC1 (Mildvan and Cohn, 1965). Ca2+ has been implicated in glycolytic control at the pyruvate kinase site in two tissues, Ehrlich ascites tumor cells and guinea pig cerebral cortex slices (Bygrave, 1966; Takagaki, 1968). Cu2+ at physiological concentrations has been ll hypothesized as an FDP—reversible inhibitor for rat and mouse liver pyruvate kinase (Passeron 35 31., 1967; Carminatti 35 31., 1968). More recent work with rat liver has shown that the FDP mediated reversal of this inhibition was of little significance (Bailey t 31., 1968; Rozengurt _; _1., 1969) . Metabolically derived inhibitors-Although ATP has gen— erally been recognized as an inhibitor of pyruvate kinases from rabbit muscle (Reynard_3§ 1., 1961), rat liver (Tanaka‘31'31., 1967; Weber 33 _1., 1967a), brewers' yeast (Haeckel _E 31., 1968), 3. xylinum (Benziman, 1969), and desert locust flight muscle (Bailey and Walker, 1969), no evidence has yet been presented that ATP may limit enzy- matic activity in glycolyzing tissue or tissue extracts. '13‘y1359, the mode of action of the nucleotide has been ascribed to the following three findings: a. competition with PEP (Reynard 31 31., 1961; Boyer, 1969). b. competition with ADP (Reynard 3; 31., 1961; Weber ._3 31., 1967a; Benziman, 1969; Holmsen and Storm, 1969). c. complexing of Mg2+ (Wood, 1969). Inhibition of brewers' yeast pyruvate kinase upon addition of citrate, NADP, AMP, 3',5'-cyclic AMP, and nucleotide triphosphates are likely to be due to the last listed mechanisr tion by 1 (Tanaka 1 Walker, L—pf sible inl ruvate k, Yielding, uterus (l rat tiSSL alanine a A cl enzyme 2E iological hibit crut as Well as heart mus, fatty acid iCiSm that inhibitioy willlaTHSOf Table he” e been 12 mechanism (Haeckel t 1., 1968). Release of ATP inhibi— tion by FDP in cooperative systems has been reported (Tanaka 35 31., 1967; Haeckel 33 31., 1968; Bailey and Walker, 1969) but the mechanism is still obscure. L-phenylalanine and diethylstilbestrol are also rever- sible inhibitors of unknown metabolic significance for py— ruvate kinase preparations from rabbit muscle (Kimberg and Yielding, 1962) and rat prostate, seminal vesicle, and uterus (Vijayvargiya t a1., 1969), respectively. For the rat tissues, L—alanine was seen to protect against L—phenyl- alanine antagonism. A class of inhibitors requiring incubation with the enzyme prior to assay has also received attention. Phys— iological concentrations of long chain free fatty acids in— hibit crude rat liver pyruvate kinase (Weber 33 31., 1967a) as well as acetyl—Coenzyme A (Weber t 31., 1967b). The rat heart muscle enzyme is also inactivated in the presence of fatty acid salts (Tsutsumi and Takenaka, 1969). The crit- icism that there has been no direct information on enzymatic inhibition 13 3133 by free fatty acids has been offered by Williamson (Weber 31 31., 1967a). 5. Kinetic cooperativity Table 1 summarizes pyruvate kinase preparations which have been noted to yield cooperative steady-state kinetics III-n .Amcmav common EOE mam mammau meQaDm umz «DQSCflUCOU» H menanLw II II +m aea .Amomav .Hm n unsmcmnom +m awn .Amwmav “0”..qu UCM HOmSm numb mmm .Aeomac meaamm can noamme mom awn .Aaomav .mm mm.mxmeme man man ne>aa new mom mom +emz .+x mom + a: .Anmomav nenaesm 6cm smamcsm mom . mam ammo» .mnexmm +~mz mam oumuuflo mom a: + I I was .Asomav .Hm be Hexummm mom can a . one + m2 +x .Aeomav meoemm can mean mom ama II II mom .Amomav .H on mmom mam ammo» .mnosoum noncommm ocmmfla oonoom oocouomom camouuououom UHm0uuoEom DE>Ncm .mommcflx oum>su%m o>flumuomooo mo mofluuomoum .H manna l4 .Amomav Owanm mam wxmuo o AOOGHV GMEH NCOm .Amomav CEMESO .Amomav ouofiow .Amomav nexnaz 6cm seaaam .Ammmac .wm wl_ceoanom .Ambmav anamofl>onsw one cmeHaz .Ammmav muoncnox can fium>ooamz .Amomav Hm3cmm Dam mamas .xmomav ceamcnaaenem> new neaox .Ammmav 20mmom mam AnnoumuomEou mcflmmouoocv mam mam mam mam mam mmm mmm mmm mmm mmm mmm mmm mmm mmm mmm mam mmm mmm Es>mam Enwuouomnw>oum Eszflawm,uouomnouoo¢ mwaflomum mcoamsm oaomofi ano mnwx Soon you unsooa uuomon mmmm can uo>wa moum Chunfio mcflapcsouo “Hon mflsowuonomm ouhuounuhuo amesm comma» onomwpm umm Aooscflucoov H manna 15 for at least one substrate, metallic cofactor, or inhibitor. Two attempts at fitting such data to the Monod-Wyman-Chan- geux model of allosteric interactions (Monod _E _1., 1965) for rat liver L (Rozengurt 31.31., 1969) and brewers' yeast (Wieker._£ 31., 1969) enzymes are limited in that only the PEP, FDP, and ATP variables were considered. 6. Temperature effects 71 Literature in this area is scanty. Kayne and Suelter (1965) observed a break in the Arrhenius plot for the muscle pyruvate kinase reaction at about 16°, the metabolic sig— nificance of which, if any, is unknown. More recently, Somero and Hochachka (1968) noted temperature—dependent changes in the Km for PEP for the rainbow trout and EEEEE- tomus bernacchii (antarctic fish) muscle enzymes. Enzyme- ATP, —ADP, and —FDP interactions were independent of tem— 2+ inhibition dependent. Minima in the PEP perature, but Ca Km's were the same as the fish habitat temperatures. II. Molecular heterogeneity and regulation of enzyme levels 1. Nutritional and hormonal control During a systematic study of the levels of enzymes related to gluconeogenesis in rat liver (Krebs and Eggleston, 1965), pyruvate kinase activities were found to vary over a 10 fold range. Highest levels were associated ( a. \- 16 with high carbohydrate diet, and lowest activities with starvation or low carbohydrate regimen when gluconeogenesis is favored. This relationship was correlated with the in— sulin level of the rat (Tanaka 33 31., 1965; Weber 33 31., 1965). Enzyme levels dropped in alloxan diabetic rats, peaked to normal upon administration of insulin, and hor- monal induction was prevented by protein biosynthetic inhib— itors. More importantly, the activity in liver extracts was electrophoretically resolved into 4 peaks, one identi— cal to the muscle enzyme and the other 3 immunologically distinct and unique to liver (Tanaka 31 31., 1965), of which only the 3 liver types fluctuated with diet or insulin. The muscle type in liver is not invariant, however. Marked increases were noted in regenerating rat liver (Tanaka t 31., 1967) and in livers of rats perfused with the blood or plasma from Walker tumor bearing donors (Suda 31 31., 1968). Actinomycin S and Effluorophenylalanine inhibit the latter phenomenon. The effect of antibiotics on enzyme levels resulting from mixed sequential high carbohydrate and high protein dietary studies (Szepesi and Freedland, 1968) illustrates the complexity of the induction process. Their data were consistent with translational level control of enzyme syn- thesis during carbohydrate to protein dietary transitions, a EC ()1 r‘ I “.1 l7 and transcriptional control during the opposite transition. No data were available on isozymic variations. Induction may be independent of insulin. Liver pyruvate kinase can be induced in alloxan diabetic rats by a fructose—glycerol diet (Sillero 33 31., 1969). Further dissection of glycolytic and gluconeogenic pathways led these workers to hypothesize secondary metabolite induction. Uterine pyruvate kinase activity is increased by es— tradiol-l79 injection of ovariectomized rats. The augmen- tation is blocked by actinomycin D, 5-fluorouraci1, or cy- cloheximide (de Asfia 33 _1., 1968). A peaking has also been reported for the rat brown fat enzyme during suckling (Hahn and Greenberg, 1968). Induction is not limited to mammals. Activity is in— duced in several yeasts (Hommes, 1966; Gancedo t 31., 1967; Fernéndez _3 31., 1967), 3. 3311 K12 (Malcovati and Kornberg, 1969), and Euglena gracilis (Ohmann, 1969). The inducible activity in 3. 3311 K12 was separated from a con— stituitive enzyme and shown to be kinetically distinct. Potassium deficiency increases pyruvate kinase from wheat seedlings (Sugiyami and Goto, 1966) by an unknown process. 2. Isozymes and multiple forms Tissue—distinct forms of the enzyme have been recog- nized for many years (Table 2). Several of these forms, in 18 mCMmSUOM ofluuooaoomfl co nonmn a uo>wg I wanna: mcflmDUOM ofluuooaoomfl .Amomav mmano co mocmo m on a msonmomm umm xaamoflnmmumoumfiouno .Amomav muoncnox can Hum>ooamz moumummom m HHOU .m. I Aeneas II II. I cacao: .Amwmav .Hm um >oamflm I um>fla Gmfism I uo>flq oxflawno>fla oco .Amomav mxcomzoom pom onoeom .mocmn vapouosmonuooao m macho: uoouu Boncflmm .Amomav .wM.MI.uoNHooN Qmoaoaam ofluocmm m noumoousumnm amass I oaomsz oxHHIoHomSE oco .Amomav.wm,mw.mxmcme .mCCmn oflumuonmouuuoao a um>flq pom I saunas I wanna: .Amomav .wm mw.anenceaaea eo> I neaan Dam .Aawmav .fll.wl oumcwom mocmp ofluouonmonuomao m nMoaomnz panama / comm mfiuom occonomom mmpfiocomououom bananas: Emficmmuo .moE>Nomfl o>flumfismonm can ommcHx oum>ouhm mo mEuom mamfluasz .m wanna 19 .ooaooum osmmfiu mason .moewnomfi o>flumESmmnm moansaocfl .cofiumsuflm ham Baum mcfluHSmmmM I unmom I no>flq I ,oaomnz nonmn ofluouonmouuooao m mmmo ponfiafluuom mpcmn cauouocmouuooao v cooamm mpcmn ofluouonmouuooao m mospflx .Ammmav .flm mm.cooH:om nonmn caumuosmonuooao m mesa moum Aomhu my mCMmSUOM oflnuooaoomfi co mccnn N noofiq mcflwooom .Ammmav .flm.mm.n0msm oflnuooaooma so mocmn o manna: Dannmm reaaaanaooc a wanes IV: 7 NH , I v fins! 90% Udlnflvflvanvfleomid f-AU fl-uVFI—flsflfl q “flux/Hal? I @HUWHHS . UCHTCUOW UHHDUOHQODH NH .7 . . um AmeHV mmHtHHV co nailing} \1 \III I tin-1)]. “my!“ 20 turn, show isozymic heterogeniety, some of which may be re- 1ated to other tissue-distinct enzymes. Regulation of in— dividual presumptive isozymic variants has not been re- ported, though variable sensitivity of purified rat liver pyruvate kinases toward Cu2+ inactivation (Carminatti ‘_3 31., 1968) and FDP activation (Susor and Rutter, 1968) might be explained in this way. 3. Conformers and interconvertibility of forms A body of literature now provides evidence for the ki- netic or physical identification of conformational states of pyruvate kinase preparations. The conversion processes are not well understood, not always reversible, and of un- certain 13 y133 importance. Crystalline rabbit muscle pyruvate kinase changed in electrophoretic mobility and limiting viscosity on the re— versible binding of diethylstilbestrol, a surface—active synthetic estrogen (Kimberg and Yielding, 1962). With the same enzyme, breaks in Arrhenius plots and temperature in— duced UV difference spectra were fitted to a two conformational equilibrium model (Kayne and Suelter, 1965). Fluorescence polarization and sedimentation parameters were consistent 'With the low temperature and cationeactivated forms being more compact than the high temperature form (Kayne and Suelter, 1968) . n- 21 Some preparations of the rat liver enzyme (Tanaka 33 .31., 1967; Susor and Rutter, 1968) are variably sensitive to FDP activation and loss of this property could be in- duced through incubation at 370 in dilute solutions or stor- age at -200 with no change in immunological specificity in the first case. On the other hand, the purified enzyme may gain sensitivity toward PEP and FDP upon purification or in— cubation at 25° (Susor and Rutter, 1968; Bailey 33 31., 1968). The latter investigators found FDP and high pH in- cubations favored the formation of a cooperative, sensitive conformation. With a more stable preparation, the facile reversible transformation of this enzyme from a cooperative, FDP-sensitive form to a normal, FDP insensitive form.was mediated by the pH of the reaction mixture (Rozengurt 33 .31., 1969). This group discovered dithiothreitol and sub— strates to protect against inactivation during purification. A similar equilibrium for rat epididymal fat pyruvate ki- nase (Pogson, 1968)‘was mediated by citrate or EDTA and countered by FDP. In this case, the conformations were not only stable enough to be electrophoretically resolved but a difference in sedimentation value was also seen for the two types. Thus, the conversions may have been associ- ation-dissociation phenomena dependent on some divalent cation. 22 Several enzymes diSplay complex pH optima with two or more peaks which may represent the activities of a mixture of conformers in the assay solution (Bailey _3 _1., 1968; Haeckel 33 31., 1968; Hunsley and Suelter, 1969b; Bailey and Walker, 1969). In the last 3 cases (yeast and desert locust flight muscle enzymes) activity peaks in the high pH range are produced upon addition of PEP or FDP, reSpective- 1y. For Alaskan king crab muscle pyruvate kinase, lower temperatures mimic the heterotropic effect of FDP on PEP kinetics common to other cooperative systems (Somero, 1969). The kinetics of the FDP-promoted dissociation of yeast pyruvate kinase are consistent with the existence in solu— tion of two conformers (Kuczenski and Suelter, 1970). METHODS AND MATERIALS I. Enzymes The pyruvate kinase in this study was purified from fresh commercial bakers' yeast, Saccharomyces cerevisiae ("Budweiser," Anheuser-Busch, Inc.), according to the method of Hunsley and Suelter (1969a) (Fraction VII), and stored at 40 as a concentrated suspension in 3.6 M (NH4)ZSO4 contain— ing 10 mM Na phOSphate buffer, pH 6.5. The preparations were stable for months in this state with no known deterior— ation in either chemical or physical properties. The mini- mum Specific activity of any preparation used, except in Mg2+ kinetic experiments (150 pmoles/min/mg minimum), was 200 pmoles of pyruvate formed/min/mg under maximal assay conditions. (NH4)ZSO4-free solutions of enzyme were pre- ,pared by Sephadex G—25 chromatography in apprOpriate buffers and tested for sulfate content with saturated BaClZ. All rmanipulations of the enzyme were at room temperature to avoid cold denaturation. For comparative purposes, phenylmethanesulfonyl fluo— ride, a potent protease inhibitor, was included in the iso- latnion procedure at the cell lysis and first (NH4)ZSO4 23 24 precipitation steps (Hunsley and Suelter, 1969a). A final concentration of 2.0 mM in the extract was used which has been shown sufficient to inhibit esterolytic protease activ— ity during the isolation of yeast hexokinase (Schulze and Colowick, 1969). No effect on pyruvate kinase activity in the extracts was noted upon the additions of inhibitor. Lactic dehydrogenase, the assay coupling enzyme, was either Calbiochem grade A or the Sigma type II rabbit mus- cle enzyme substantially free of pyruvate kinase and was desalted free of (NH4)ZSO4 over medium Sephadex G—25 in 0.20 M Tris-HCl, pH 7.5, before use. Rabbit muscle pyruvate kinase used in the assay of PEP and ADP was prepared by a modification (Kayne and Suelter, 1965) of the Tietz and Ochoa (1958) procedure. The following enzymes and proteins were used as molec— ‘ular weight standards: crystalline bovine serum albumin, crystalline rabbit muscle aldolase, 3 times recrystallized soy bean trypsin inhibitor, crystalline swine stomach pep— sinogen, equine alcohol dehydrogenase, 5 times recrystal— lized bovine pancreas ribonuclease, crystalline pig heart ma lic dehydrogenase, type II oC—chymotrypsinogen A (all Sigma products), and Sperm whale myoglobin (Calbiochem A grade). Mixed crystals of rabbit muscle a(—glycer0phOSphate dehydrogenase and triose phOSphate isomerase were from . Cu 1( CE ti ”- yC 5‘, ‘L1 HUI ic 25 Calbiochem. II. Chemicals All water was either double glass distilled or glass distilled and deionized (Crystalab Deeminite). Pyruvic ac— id (Matheson, Coleman, and Bell), distilled at reduced pres— sure, was neutralized with (CH3)4NOH. TriCHA PEP, Tris ADP, Na ADP, tetraCHA FDP, Ba FDP, and Na NADH were Sigma Chemi- 14C, specific activity cal Company products. Ammonium FDP- 69 mC/mM, was purchased from the Amersham—Searle Corpora- tion. Ba FDP was converted to the (CH3)4N+ salt with [(CH3)4N] 2804 prepared from Eastman (CH3)4NOH. Na ADP rou— tinely was chromatographed at pH 7 over Dowex 50W-X8, Tris cationic form, to yield the Tris salt. Eastman (CH NCl was recrystallized from hot ethanol 3)4 (Kayne, 1966). Sephadex G-25 (medium), G—200, and Blue Dex- tran were from Pharmacia. N, N, N', N'-tetramethylethylene— ciiamine, N, N'-methylenebisacrylamide, and acrylamide were supplied by Canalco and used without further purification. chty per cent solutions of Ampholine carrier ampholytes, 1&1 ranges 3-10 and 5-8, were LKB products. All other chem- icals and reagents were of the highest commercially obtain— able purity. 26 III. Experimental methods 1. Assay of activity and determination of enzyme concentration After desalting over Sephadex G—25 in H20, an extinc- tion coefficient at 280 nm was determined for purified en— zyme by plating approximately 5 mg aliquots of known Opti- cal density in tared vials. The samples were lyOphilized and dried to constant weight over P205 at 1100 (Hunsley and Suelter, 1969a). Standard assays were performed at 300 on dilutions of the enzyme in 50% (v/v) glycerol—10 mM Na phos— phate buffer, pH 6.5, by employing the linked lactic dehy— drogenase reaction modified from Bficher and Pfleiderer (1955). The reaction mixture contained, per m1: 100 pmoles (CH3)4N cacodylate, pH 6.2; 24 pmoles MgClZ; 100 pmoles KCl; 5.0 pmoles triCHA PEP; 10 pmoles Tris ADP, pH 7; 1.0 pmole tetraCHA FDP; 0.15 pmole Na NADH; and 33 pg (NH4)ZSO4—free lactic dehydrogenase (Hunsley and Suelter, 1969a). Routine assays in the absence of FDP were identical except for an additional 100 pmoles KCl per ml and omission of FDP. Ali- quots of 10 pl or less of enzyme dilutions were added to the reaction mixture at 30° in a 1 cm silica cuvette and the change in optical density at 340 nm was recorded on a Gil— ford model 2000 modified Beckman DU ultraviolet spectro— photometer. The initial rate was converted to micromoles of 27 pyruvate formed per minute by dividing the change in Optical density per minute by 6.22 (Horecker and Kornberg, 1948). ADP and PEP concentrations were estimated by a modifi— cation of the Bficher and Pfleiderer (1955) pyruvate kinase assay in the presence of excess rabbit muscle enzyme. FDP was estimated in the presence of rabbit muscle.(-glycero- phOSphate dehydrogenase, triose phOSphate isomerase and ex— cess aldolase as modified from the assay of Rutter 33 31. (1966). 2. Electrophoresis and chromatography Disc gel electrophoresis—50 pg of enzyme was electro— phoresed in 6.0% polyacrylamide gel at 250 in 5 X 75 mm columns (Davis, 1964), stacking at pH 8.3 and running at pH 9.5. No sample gel was used. Instead, the samples were applied in 50%I(v/v) glycerol containing 15 mM Tris phos- phate, pH 6.9. After staining with 0.55%.Amido-Schwarz in 7.5%.acetic acid, the gels were destained electrOphoretical- 1y. Gel chromatogrgphy-An analytical Sephadex G—200 column, 2.6 X 36 cm, was poured with a 1.8 cm overlay of G-25. A hydrostatic head of 10 cm was maintained for the 0.10 M Tris-HCl, pH 7.5, elution buffer. One mg samples of protein were added to the column in 1.0 ml of 0.50 M sucrose—0.10 M Tris-HCl, pH 7.5. Effluents were automatically monitored at 28 235 nm in a Beckman DB ultraviolet spectrophotometer fitted with a flow cell, and fractions collected with a calibrated drop counting fraction collector. The void volume was de- termined with 0.2%IBlue Dextran added in Tris—sucrose buf- fer (Andrews, 1965). 3. Chemical prOperties Metal content—(NH4)ZSO4—free solutions of the enzyme in 0.10 M Tris—HCl, pH 7.5, were analyzed for metal content. Samples were examined on either an Aztec or Perkin—Elmer atomic absorption Spectrophotometer. In addition, approx- imately 11 mg aliquots were dried on Whatman No. 54 acid washed filter paper discs, 2.5 cm diameter, and examined in a General Electric XRD—6 x—ray fluorescence unit. Secondary standards of CaClz and MnC12 were plated on identical filter discs. Amino acid analysis—The enzyme was prepared for hydrol- ysis by desalting on a G-25 Sephadex column in H20. The samples were hydrolyzed in constant boiling HCl according to Bailey (1967). Amino acid analyses were performed by the method of Spackman 33 31 (1958) on a Beckman—Spinco model 1203 amino acid analyzer and by the Piez and Morris (1960) modification of this procedure. Combined cysteine and cys- tine were estimated after performic acid oxidation (Moore, 1963) and hydrolysis, and determined as cysteic acid. ’t’ >1 29 Tryptophan was estimated on samples desalted over Sephadex G—25 in 0.10 M Tris-HCl, pH 7.5, according to Goodwin and Morton (1946) with precautions noted by Beaven and Holiday (1952). Spectra were obtained with a Beckman DB ultraviolet spectrophotometer (Hunsley and Suelter, 1969a). 4. Kinetic properties Linked assays as described previously were conducted at 300 on enzyme dilutions in 50% (v/v) glycerol—10 mM Na phos— phate, pH 6.5. Extraneous alkali and ammonium ions in ki— netic experiments were estimated to be 300 pM in Na+ from NADH additions and less than 100 pM in NH4+ from enzyme ad- ditions (Hunsley and Suelter, 1969b). The reaction was ini- tiated in all experiments by addition of enzyme. In addi- tion, all catalytic variables except the one or two under examination were at saturating or near saturating levels. All rates were corrected for any blank rate encountered a- rising from pyruvate kinase in the lactic dehydrogenase. In studies involving measurement of pH, the reaction mixtures 'were tested directly immediately after assay with a Sargent :model LS pH meter fitted with a Sargent S—30070-1O unit electrode. Where kinetic data are treated as Hill plots, total concentrations of added substrate or activator are found on the abscissa. Lines through the points were drawn by eye 30 and the values of the slopes do not indicate the limits of accuracy of the experiment. The apparent Km or KA is de- fined as that concentration of substrate or activator where v = % maximal observed velocity. The Hill slope, nH, is defined as Alog (v/V—v)/Alog [L] where v is the observed velocity, V the maximal observed velocity, and L the vari— able ligand (Atkinson, 1966). 5. Binding properties The binding of Mn2+ to yeast pyruvate kinase was stud— ied at 300 by electron paramagnetic resonance and proton re- laxation rate measurements as previously described for the rabbit muscle enzyme (Mildvan and Cohn, 1965). The data were plotted according to Scatchard (1949). The interaction of substrates with the enzyme—Mn complex was studied by the proton relaxation rate method and the data were treated by procedures I and III of Mildvan and Cohn (1966). Binding of FDP to the enzyme was tested by incubation of the enzyme with FDP—14C, Sephadex G—25 chromatography of the mixture, and identification of effluent protein and radioactive peaks. Radioactivity was monitored by scintillation counting in a Packard Tri—Carb model 3310 scintillation SpectrOphoto- meter with a window setting of 50-1000 at 15% gain. Known 14 aliquots of FDP— C were used as standards. The 31 scintillation mixture (Gordon and Wolfe, 1960; Kinard, 1957) contained 4%.Cab-O-Si1. 6. Isoelectric focusing Preparative procedure-A 110 ml capacity LKB model 8101 electrofocusing column was loaded with a linear 20-60% (v/v) glycerol gradient containing 1% final concentration of pH range 5-8 carrier ampholytes titrated to pH 7.2 with (CH3)4NOH. The voltage was raised in 100 v steps to 700 v F ""9 in 3 hours and maintained there at 200 for 72 hours, after which time the column volume was diSplaced with H20 and fractions collected (Vesterberg and Svensson, 1966). Pro— tein concentrations at the termination of the experiment were estimated by a nephlometric assay (Mejbaum—Katzenellen— bogen and Dobryszycka, 1959) with yeast pyruvate kinase standards. Microisoelectric focusing 13 polyacrylamide gels-Elec- trofocusing was performed at 25° (Catsimpoolas, 1968) in riboflavin-catalyzed 6.5% polyacrylamide containing 2% carrier ampholytes (pH range 3—10) titrated to pH 7.2 with (CH3)4NOH. 5 X 75 mm glass columns were coated with a so- lution of 5%IPlexiglass in CH2C12 and dried prior to use to facilitate removal of gels from the columns (Loening, 1967). Riboflavin—catalyzed gels were polymerized for 30 minutes 3 32 inches from a fluorescent lamp. 4.5% polyacrylamide gels were polymerized 15 minutes with 0.054% ammonium persulfate in the light. Cathodic ends of gels were marked with pow- dered charcoal for easy identification. All columns were focused at 5 ma per tube initial current in a disc electro- phoresis apparatus with 5% phosphoric acid in the anode bath and 5% ethanolamine as the cathode. After focusing, gels were removed from columns with wa— ter from a long blunt No. 22 hypodermic needle, fixed one hour in 10% trichloroacetic acid, and then transferred to 7% acetic acid for storage. To quantitate precipitated enzyme bands, gels were placed in 0.5 X l X 10 cm silica boats un— der 7% acetic acid and scanned at 280 nm in the Gilford lin- ear transport apparatus. Scan tracings were cut out and weighed. Since carrier ampholytes interfere strongly with pro- tein dyes and dialyze out only very slowly, visualization of the protein was accomplished by light scattering. Gels were placed in 7 X 125 mm glass tubes which were filled with 7% acetic acid, and the tubes corked. These were placed in a black—bottomed Plexiglass tray, covered with ethanol, and illuminated along the axis of the tubes by a 150 watt re— flector flood bulb through two successive 5 X 100 mm slits. Photography was accomplished at 900 to the incident light in a darkened room. 33 RESULTS I. Criteria for purity 1. Disc gel electrOphoresis One of the most sensitive techniques available to the enzymologist for physical separation of protein species is polyacrylamide disc gel electrophoresis which differentiates by both charge and size (Ornstein, 1964). To establish that the purified yeast pyruvate kinase under investigation was molecularly homogeneous, enzyme at all stages of purifica— tion was routinely electrophoresed by this method. The enzyme sample was placed directly on the spacer gel in glyc- erol-buffer solution which protects against cold—induced in- activation (Hunsley and Suelter, 1969a) and subsequent pro- duction of complex mixtures of dissociated enzyme (Kuczenski and Suelter, 1970). Illustrated in Figure l are the results of a disc electrOphoretic experiment which demonstrated coincidence of enzymatic activity and the major stained protein band. The minor constituents comprised less than 5% of the total pro- tein in the final yeast pyruvate kinase product and can be eliminated by narrowing the pool width of the last 34 35 chromatographic purification step (Hunsley and Suelter, 1969a). The pyruvate kinase stained disc migrated as a sin- glet independent of the amount of protein, the gel concentra— tion, or the addition to polyacrylamide gels of reagents which are known to stabilize enzymatic activity: either 1 mM tetraCHA FDP and 10 mM MgC12 or 12.5% glycerol. 2. Contaminating enzyme activities Two enzymes which could, if present in significant amounts as contaminants in the pyruvate kinase preparations, interfere with kinetic and binding determinations are adeny- late kinase (2ADP=—'—‘AMP + ATP) and aldolase (FDPr—‘glycer— aldehyde-3-ph08phate + dihydroxyacetone phOSphate). Adeny- late kinase activity in the purified product, assayed with a hexokinase-glucose-6—phosphate dehydrogenase—linked reac- tion in the presence of ADP, glucose, and NADP, was less than 2 X 10"3 pmoles/min/mg, the lower limit of the deter- mination. Since yeast aldolase has been shown to be ex- tremely unstable in the absence of sulfhydryl-reducing agents, it may be ignored (Rutter 33 31., 1966). II. Chemical properties 1. Extinction coefficient The dry weight based extinction coefficient at 280 nm, Eg-g = 0.653, lower than that defined by the Warburg and 36 Figure 1. Disc gel electrophoresis of 50 pg of yeast py- ruvate kinase. The enzyme was electrophoresed as outlined in Methods and Materials in 6.0% polyacrylamide gel. The gel was split longitudinally, one half stained, and the other sliced into two mm pieces and each piece diSpersed in 0.10 ml of 50% glycerol—10 mM Na phosphate (v/v), pH 6.5. An aliquot of 10 pl of this suspension was tested for ac- tivity in the standard assay containing FDP. The enzymatic activity was coincident with the dark staining band near the left cathodic end. The band to the extreme left was the opaque spacer gel (Hunsley and Suelter, 1969a). 37 Activity (units/2 mm. slice) IO 20 30 40 50 mm. from origin Figure l 38 Christian (1942) assay, reflects the relatively low aromatic amino acid content of the enzyme (Hunsley and Suelter, 1969a). 2. Amino acid analysis The amino acid composition (Table 3) is the average of two independent analyses. Uncertainties in extrapolation to zero time for threonine and serine residues gave rise to comparatively larger standard deviations. Determination of amide nitrogen of aSparagine and glutamine was beyond the scope of this investigation and, therefore, aSpartic and glutamic acid values represent combined acid and amide resi- dues. The value found for NH was 103 t 1.2 moles per 3 165,800 g of enzyme. Alternate data for tyrosine are avail- able from the alkaline ultraviolet absorption tryptOphan estimation of Goodwin and Morton (1946)o Forty-seven tyro- sines per 165,800 g of enzyme (Hunsley and Suelter, 1969a) were estimated by this less reliable method. 3. Metal content The NMR proton relaxation rate studies reported in this thesis are based on the observation that paramagnetic ions greatly increase the relaxation rate of nuclear spin states of water protons (Cohn and Leigh, 1962). During preliminary 2 Mn + binding studies, relaxation rates of water protons in buffer containing only enzyme were unusually high. 39 Table 3. Amino acid analysis of yeast pyruvate kinase. Methods of analysis are described in Methods and Materials. The results are expressed as the mean 1 standard deviation of two determinations (Hunsley and Suelter, 1969a). Amino acid Moles/165,800 g enzyme Lysine 99.4 i 2.0. Histidine 21.2 i 1.9 Arginine 64.8 i 1.2 Aspartic acida 156.9 i 7.8 Glutamic acida 109.1 i 2.2 Proline .68.6 i 1.5 Glycine 94.4 i 2.2 Alanine 122.5 f 2.2 Valine 113.4 f 5.3 Methionine 22.0 i 3.4 Isoleucine 82.6 i 5.2 Leucine 106.0 1 1.3 Tyrosine 38.1 i 1.1 Phenylalanine 43.2 i 0.3 Cysteine 14.4 i 0.3 Threoninec 178 i 15 Serinec 113.9 1 8.3 Tryptophan 9.3 i 0.2 ‘EAcid and amide conbined. etermined as cysteic acid. cValues extrapolated to zero time hydrolysis. 40 Contaminating metal ions such as Cu or Fe were suspected and the enzyme purification was modified so that the source of the metals, diatomaceous earth, was free of such ions. Ex— amination of the modified preparation by atomic absorption Spectrophotometry (Table 4) revealed Zn, Co, and Fe content to be reduced below the limit of detection for each. Cu content remained constant, regardless of whether enzyme so- lutions were treated with Chelex chelating resin or EDTA was incorporated in each step of the enzyme purification. Table 4. Metal content of purified yeast pyruvate kinase. Purified enzyme was desalted in 0.10 M Tris-HCl, pH 7.5, over Sephadex G-25 and examined by atomic absorption Spec- trOphotometry as described in Methods and Materials (Hunsley and Suelter, 1969a). Metal Moles metal/165,800 g enzyme Zn <0.01 Co <0.0l Fe <0.05 Cu 0.14 No other unexpected elements in dried enzyme samples ‘were found at levels greater than 0.01 moles per 165,800 g of enzyme through the use of an x—ray fluorescence unit, scanning from atomic numbers 13 through 30 (Hunsley and Suelter, 1969a). 41 4. Gel chromatography Analytical gel chromatography proved not to be a useful tool for analysis of the molecular weight of pyruvate ki— nase. The proteins used as standards behaved as typical gldbular proteins with a molecular weight dependent elution volume (Andrews, 1965), but yeast pyruvate kinase eluted as skewed, non—Gaussian peaks correSponding to a molecular weight of up to 300,000. Activity was distributed through— out the peaks. III° Kinetic prOperties 1. Requirement of monovalent cation The presence of Specific activating monovalent cation was required for Optimum catalytic function of yeast pyru— vate kinase (Table 5). (CH3)4N+ ion could not replace alka— 1i metal or ammonium ions demonstrating that the effect was not merely due to increased ionic strength. Sodium ion pre- sented an intermediate case, that of functioning only in the presence of FDP, the allosteric activator. The very low rates associated with FDP addition alone and in combination with (CH NCl can be explained, then, through potentiation 3)4 of low contaminant Na+ levels in the assays. Serial transfer of a clone of the original yeast was made in aerobic complex growth medium containing increasing concentrations of KCl up to 2.7 M. Growth at high salt 42 Table 5. Requirement of yeast pyruvate kinase activity for alkali metal or ammonium ions. The assay mixture (1.00 ml) contained 100 pmoles (CH3)4N cacodylate, pH 6.2, 24 pmoles MgClz, 5.0 pmoles triCHA PEP, 10 pmoles Tris ADP, 33 pg lactic dehydrogenase and 0.15 pmole Na NADH. FDP was added as the (CH3)4N salt (Hunsley and Suelter, 1969b). Reagents Final Initial velocities added concentration (mM) (pmoles/min/mg) FDP 1.0 1.5 (CH3)4NC1 50 4 0.10 (CH3)4NC1 200 Iflca .momonucoumm ca csocm mum mo>noo ecu you momon Adam .mncz mz mace: ma.o pom .ommcomouomsmp ofluoma mm mm .Aucommum co£3v mam Zeammov mace: o.H .m04 mane moaofil OH .mmm «Evan» mma081 o.m .maomz moaofim on .m.© mm .oumamcoomo Zafimmov moHoEA ooa oocamucoo AHE oo.Hv ououxfle momma one .coflumuucoocoo coflumo ucoam>fico mcflum>fluom can mmmcflx oum>oumm unmom mo muflooamo Hmauflcfl coosuon mwsmcoflumaom .N ousmflm 45 :5 r2 8.. ears: 8.. on- «a- e..- no. on- «a- a... mo- . .I q _ . ,_ o. d a, _ . _ q _ teen. s... 3.2 o Ir .3... 53 t. o I 5.. _l .7'12 8 0 1r +x 0 l 1. ea... 53 +az _. .. o. _- 0. O C) - J}. (I AI 9m ‘9. {N Figure 2 46 reduced. To summarize, FDP, in the case of monovalent cat- ion, not only reduced cooperativity nearly to classical Michaelis-Menten kinetics (nH = 1.00) but also decreased the apparent KA. This pattern of FDP activation was seen for all remaining variables except ADP. The activation by FDP in the presence of low concen- trations of the required monovalent cations K+ and NH4+, was also cooperative with nH equal to 2.33 and 2.55, re— spectively. Na+-dependent FDP activation gave a Hill Slope near unity which was unaffected on lowering the Na+ concen— tration (Figure 3). Similarly to monovalent cation kinetics, with PEP as a variable (Figure 4) the homotropic effect was effectively abolished by the addition of FDP and the apparent Km's lowered by an order of magnitude or more. This same rela— tionship held true for M92+ (Figure 5), although c00pera- tive behavior was still evident with FDP present. The Hill slope for the assays containing Na+ remained intermediate in this instance at 2.86. Only a very small FDP heterotropic effect on ADP ki- tnemics was seen (Figure 6). This substrate displayed laichaeliS—Menten kinetic behavior regardless of the pres— Ience or absence of FDP at saturating levels of PEP and di- valent and monovalent cations. 47 Figure 3. Relationship between initial velocity of yeast pyruvate kinase and total FDP concentration. The assay mix- ture (1.00 ml) contained 100 pmoles (CH3)4N cacodylate, pH 6.2; 24 pmoles MgClz; 5.0 pmoles triCHA PEP; 10 pmoles Tris ADP; 33 pg lactic dehydrogenase; 0.15 pmole Na NADH; and o—o, 140 pmoles NaCl, H, 10 pmoles KCl, and H, 5.0 pmoles NH Cl. Hill slopes for the curves are shown in pa- rentheses. FDP was added as the (CH3)4N+ salt (Hunsley and Suelter, 1969b). 48 2.0 I l I l I I r I V l l ' a Na“ dependent activation '5 " a K" dependent activation , 2 _ 0 NH.‘ dependent activation LOG (‘7’;4) O l l 1 l 1 l 1 l 1 - 1 1 l I462.2 -2.6 -3.0 -3.4 ~33 -4.2 LOG [FDP] (M) Figure 3 49 .Anmoaa .nonneam one monetary Damn «moan» any mm cocoa mm3 mmm .nomonuconmm a“ czocm mum mo>uoo on» now momon HHflm #0va moaoal om . OIIO com Hovmz moaofil cm .I .wlmmuflluu can “.894 moaofil o: .DIIID pom .Hox moHOei OOH .OIIIO 78v“ moHoel oma .I 5on $542 94 mace—1 mH.o “anacomoupwsoo opp IUMH 01 mm “Accomoum coczv mam Zeammov maDEi o.H “mam mane mmH081 OH “Naomz moaofil am am.o mm .oumawooomo Zafimmov moaoal ooa tonamucoo AHE oo.Hv onouxfia momma one .coflumnucoocoo mmm Hmuou can ommcflx oum>ou>m ammo» mo muflooao> Haauflcfl coo3umn mangOADmHom .v apnoea 50 22v Emma 00-. 22V ”dung 00-. Nd- ¢.¢- Qn- QN- QN- Nm- ¢.¢- Qm- QNI ON- : a — q _ In d In _ a _ SI _ a _ a _ J} O O O .- mcm SE! +¢Izo it do“. 5:3 +v. o one gm. _- - +¢IZ o no in +v. a U I II no... 2:3 +02 a I QC- . -. . m 9 1 ll l o ) i_A _I IT. I. — O . . . | l o 80 a 3m NV II 30 9 1 $6 I.\ (D Figure 4 51 .Apmoma .Hmuaosm pom woflmcsmv mononucoumm Ca c3onm mum mo>uso on» How moccam HHflm .Hovmz mmaOE: om .010 one .352 egos: om .I damm- ocm h34.2 moses: o: .ul-o one .Hos moses: 00H .1 .Hox moaoal omH . I .umoa 59% .24 mo oaofil mad “camcomonpmsop cauoma m1 mm “Aucmmoum coc3v mom Zaammov oHOE1 o.H “mam mane moHoEA OH ammo «Sofia» moHOEl o.m am.o ma .oumazpoomo Zaammuv moaofil ooa oocflmucoo AHE co.HV onsuxflfi >mmmm 0:9 .coflumuucoocoo «Hum: Howey pcm ommcfix oum>su>m Dame» mo muflooHo> HMMDHCA cooBDoQ mfismcoflumaom .m ousmwm 52 es {ea 03 12:38; 8.. ¢.n- on- QNI Nu. m... w..- o..- tn- 0.». od- md- m..- a..- o..- . a _ 1 a . . a _ a _ . _ a _ a _ . _ . a . _ 7 so. I II o .10;- O I O O J D I. r J .1N4- f ooo o I o o I O o o a I .I oo. _- fiI- a I I I .Ide t I o- 1- 1 I I o O I I I ha I I to )A AOhéo I I A_ I I no ( I i ll 1 - — . e . II 383.8 I . r . e9. 93 t. o o . fl ll U.~°’:0° +x 0 l4 O.N v I 3.2500 do... ago + :2 o I- “.2053 I I 38386 +vzz . II . do... can +02 a I IN — p b _ _ FF p — s _i p p h p p n b p F b — p p - Figure 5 53 .Anmmma .uouaosm can moamcsmv Damn maps on» no catch mm3 mam .momonucoumm cfl c3ocm mum mo>uso on» now momoam Haflm .Hoamz hence; om .ol-o one Samz hence; em .I dude-m. one “6,2,3 8621 can . nTIa one Joe moH081 OOH .AYIIO_.HOX moaofil oma .OIIIO..umoH “mamz oz oaofil mH.o nommcomonpmsop UHuUmH on mm “Aoeemena cease eon ZeAmmoc woos; 0.3 home arcane hence: o.m “mace: hoses; em "~.o mm .oumawpoomo zvammov moHoEA OOH cocflmucoo AHE oo.Hv mucuxfiE woman one .coflumuucoocoo mam HMDOD can ommcwx mum>su>m ammo» mo wuflooHo> Hafiuflcfl cooBqu mflnmcoflumaom .o ousmflm 54 25 $03 60-. as was 8.. ON- m..- m.¢- od- o.¢- 0.». Nu- m.- ed. o.- of. we- 0.? mm- ~.m- ad. ad... Qu- aujldldiqdd—ddS-_a —i4_d-d-14qi. merges eon as. .312 o .. 32:8 .3... as t. o . 2.82.3 +vxz . II 3853 +x . o I n hrb 1 1Lul "L 332.8 e9. Ba .62 .. N..- 6.0- ad- C) Q: C) ca 0 N; m.— -J‘; (I A) 90'! Figure 6 55 With all substrates saturating, FDP had no effect with- in experimental error on the observed maximal velocity of the reaction with K+ or NH4+ as the activating monovalent cation (Figure 7). An analogous experiment was not possible for Na+ since no activity with this ion is discernible in the absence of FDP (Table 5). In Figure 8 are plotted the pH profiles of the K+, K1— FDP, and Na+-FDP systems at variable PEP levels. Activity fell off rapidly on the acidic limb of the curves. The basic sides displayed complex profiles with discernible reproducible shoulders. In addition, the inclusion of FDP at low concentrations of PEP broadened the maxima in the basic pH range. 3. Kinetics of the Mn2+ activated system pH 7.5 was chosen for the majority of these experi— ments because the salt-free enzyme had maximal stability at room temperature in this range. Concentrated solutions of the enzyme in 0.10 M Tris-HCl, pH 7.5, after 3 hours at 250 retained identical PEP and FDP kinetic parameters with a loss of total activity of about 10%. Binding experiments reported later in this thesis required such stability. At pH'S below 7 the enzyme was very labile, even at room tem- perature. Figure 9 illustrates the effect of varying Na+ or K+ 56 .Anmomn .uouaoom can hoamcomv mcmz mz mace: mH.o pom .ommcomonp%soo ofiuoma mm mm .Aucomoum cos3v eon Zexmmoc hence; o.m .eoe mane moses; NH .ame «moan» hence: NH .maomz hence1 an .m.@ a .wumahpoomo szmmUv moHofil 00H cocfimucoo AHE 00-Hv onouxfifi momma one Ho mz no HUM can ommcflx oum>summ ammo» mo mufiooam> Hmfiuflcw coo3uon mflsmcoflumaom - mCOH. u. MHUCOUCOU .h ouomflm 57 Asc=o¢12_ 2): =3: moo woo moo moo _oo o 8.0 so so 8.0 cod 0 . a _ _ _ _ _ a _ _ o 1.. . . ea... 53.. .e an... 53.. .. ea... 9:. .. ea... 8. w s I an J00 00. On. (Ow/quI/salowriM. Figure 7 58 Figure 8. The effect of pH and PEP concentration on the ac- tivity of yeast pyruvate kinase. The assay mixture (1.00 ml) contained 100 pmoles Tris acetate buffer, 24 pmoles MgClz, 10 pmoles Tris ADP, 33 pg lactic dehydrogenase, and 0.15 pmole Na NADH. In addition, the top curves contained 170 pmoles NaCl and 1.0 pmole tetraCHA FDP, the middle curves 100 pmoles KCl and 1.0 pmole tetraCHA FDP, and the bottom curves 180 pmoles KCl; o——43, 10 pmoles triCHA PEP; H, 1.0 pmole triCHA PEP; H, 0.50 pmole triCHA PEP; IF—dl, 0.10 pmole triCHA PEP; and VL—47, 0.30 pmole triCHA PEP (Hunsley and Suelter, 1969b). 59 I 25- E3 5 20 I?) I- I5 I! 22 IO > 5 O IZOh- 8 loo- 5 t?) so ’2 g 60-- > 40- 20- O IZOP- 8 - :5 IOO I?) t: 80" g 60- > ‘40- 20- O, 41) Figure 8 uh I ' l Na‘ and FDP -_ ‘ K1’ and FDP 1 «shown octivatod " q .L 0 A l I ' I K "’ activated 8.0 90 60 Figure 9. Relationship between initial velocity of yeast pyruvate kinase and NaCl or KCl concentration. The assay (1.00 ml) contained 100 pmoles Tris—HCl, pH 7.5, 1.0 pmole MnClZ, 1.0 pmole triCHA PEP, 1.0 pmole Tris ADP, 1.0 pmole tetraCHA FDP (when present), 33 pg lactic dehydrogenase, and 0.15 pmole Na NADH. Hill slopes for the curves are shown in parentheses. 61 I 2 I l I l I l I l I l I | w 0.8- / - ' I. / .- A 0.4— '1 T _ (as/fl _ {'3’ o _ — 0 - .. O -' -o.4- — .. D Na"b with FOP .. -03- ' K+ _ _u 0 0 K1 with FDP _ . , -| .2 t l 1 l I I l l I l l i -O.8 -l .2 -| .6 -2.0 -2.4 -2.8 -3.2 LOG [W] (M) Figure 9 62 concentration on pyruvate kinase catalysis. Here, in con- trast to the Mgz+ system (Figure 2), FDP neither greatly decreased the cooperativity of K+ activation nor affected binding. Hill slopes of less than 1.00, observed for Na+ (0.81) and K+ (0.78) in the presence of FDP, are indicative of negative cooperative phenomena (Levitzki and Koshland, 1969). Since very small velocity changes could be noted upon addition of FDP to the kinetic system containing K+, an experiment similar to that of Figure 3 could be performed only with Na+ (Figure 10). In this case the FDP Hill lepe was 1.20 indicating nearly linear kinetics. The familiar heterotropic FDP activation of PEP and M92+ held true for the Mn2+ system (Figures ll-13). The reSponse of the enzyme towards Mn2+ at pH 7.5 and 6.2 2+ at (Figures 12 and 13) closely resembled that towards Mg pH 6.2 (Figure 5), with the exception of the Na+-dependent kinetics (Figure 13) which suggest a pH dependent enzyme—Mn interaction. Again, under saturating conditions, little effect of FDP on ADP kinetics was observed (Figure 14). 4. Na-FDP kinetic interactions Since, without FDP incorporation, no activity was ob— served with Na+ as the activating monovalent cation, the 63 Figure 10. Relationship between initial velocity of yeast pyruvate kinase and total FDP concentration. The assay (1.00 ml) contained 100 pmoles Tris—HCl, pH 7.5, 1.0 pmole MnClz, 200 pmoles NaCl, 1.0 pmole triCHA PEP, 1.0 pmole Tris ADP, 33 pg lactic dehydrogenase, and 0.15 pmole Na NADH. The Hill slope for the curve is shown in parentheses. FDP was added as the tetraCHA salt. 64 I I I I " ./ 0.8— o/ .. I:,/ A 0.4- (I20) I r {If 0 _ 8 - o “-1 '0-4— /a/ -O.8— "emu l I I I I I -3.8 -4.2 -4.6 -5.0 LOG [FDP] (M) Figure 10 65 Figure 11. Relationship between initial velocity of yeast pyruvate kinase and total PEP concentration. The assay (1.00 ml) contained 100 pmoles Tris—HCl, pH 7.5, 1.0 pmole MnClz, 200 pmoles NaCl or KCl, 1.0 pmole Tris ADP, 1.0 pmole tetraCHA FDP (when present), 33 pg lactic dehydrogenase, and 0.15 pmole Na NADH. Hill slopes for the curves are shown in parentheses. PEP was added as the triCHA salt. 66 l.2h- .0 m I A‘SQ n,n (D!I- -— fimr "- i. 0’}: — o o _ I "' (l.l5) >l> O b o -n V . OP/ 0 L9 I- / / A O o o -’ -0.4— 0’ " . / o III--“ with FDP .. -08- . ° 0 K+ _. __ / 0 K” with FDP _ O _| . at I. l l I l L l l l l -§.8 - 3.2 -3.6 -4.0 -4.4 -4.8 -5.2 Figure 11 LOG [PEP] (M) 67 Figure 12. Relationship between initial velocity of yeast pyruvate kinase and total MnCl concentration. The assay (1.00 ml) contained 100 pmoles Tris—HCl, pH 7.5, 200 pmoles NaCl or KCl, 1.0 pmole triCHA PEP, 1.0 pmole Tris ADP, 1.0 pmole tetraCHA FDP (when used), 33 pg lactic dehydrogenase, and 0.15 pmole Na NADH. Hill slopes for the curves are Shown in parentheses. 68 l .6 T I l l I l . ' l ' l ' l I .2 b _ . U " (3. 821/ (2 .08/ (I39) °‘ 0.8 '- -I A b O ‘ T 0.4- .D/ .. >4? - . . 8 ° - // ~ ._I . g a . D “0.4- '1 b a 4. . ., // D No wIth FDP -o.s - o . K, .- I- a o O K+ With FDP -' .2 I... 1 l l L l l l l I I l l '22 '25 '3.0 “3.4 “3.8 -4 2 '4.8 Figure 12 LOG [MI-2*] (M) 69 Figure 13. Relationship between initial velocity of yeast pyruvate kinase and total MnClZ concentration. The as— say (l.00 m1) contained 100 pmoles (CH3)4N cacodylate, pH 6.2: 5.0 pmoles triCHA PEP; 5.0 pmoles Tris ADP; 1.0 pmole tetraCHA FDP (when present); 33 pg lactic dehydrogenase: 0.15 pmole Na NADH; and H, 170 pmoles NaCl, H, 180 pmoles KC1, and o———o, 100 pmoles KCl. Hill slopes for the curves are shown in parentheses. _ ..._.. -_.._. .,_... N o |.6 I2 ’7‘ 0.8 >3 0.. 0 3 0 “0.4 “0.8 -l.2 Figure 13 7O I T I I I I ' l r / - o p.— .— [l' / ° -— D, /O o — (L43) 7 (3.82) 00 (“9) o P D a, o/ L— o/ 9 D/ . 0°C —-4 — a 9 D . O/ ——1 a :f' o/ _ Io ./ a Na’ with FOP. DD . K‘.‘ _. ”l J, c K’ with FOP. 1 . T . 1 L l . l “2.2 “3.0 “3.8 “4.6 “5.4 LOG [Mn2*] (M) 71 .uamm mane may mm omoom mm3 mam .mmmocucmumm cfl c30nm mum mm>u50 msu mom mwmon HHHE .maaz mz maofil mH.o ocm .mmmcmmou©>cm© ofiuoma mm mm .Hox no Homz mmaoal oom .Apcmmmum amaze mom «momuumu mace: o.H .mmm «mofluu macs; o.H .maocz macs: o.H .m.> ma .Humumflue moa081 ooH omamucoo AHE oo.av mucuxwe hmmmm one .cofiumuucoocoo maa Hmuou 0cm mmmcflx mum>9u>m ummwh m0 wuflooam> Hmfluflcfl cmm3umfl mfismcoflumamm .va musmflm 72 2): _Haodu 00.. 0.0- 06: Ni. QM- can- Qmu 0N- Nd- a _ _ . 4 _ a _ _ _ _ _ m. T .. mom 5:3 .L. o n 1 +X O IN._I inc... 5:: +02 a .. _l u 1 O 9 l lco: ) , o m - AA .. . \ . ... o .1 I.\ - K . :N: I l¢.o l 10.0 _ t _ b b _ F igure l4 73 sstudy of the mutual kinetic dependence of these ligands was :facilitated. Increasing concentrations of FDP proved to (decrease the Hill slope for Na+, but increase the apparent I§A° In Figure 15 are plotted the Vm's for a series of ki- rietic determinations at 5 different NaCl concentrations, (each extrapolated to infinite FDP concentration. The KA :Eor NaCl at infinite FDP concentration was 100 mM. In con- ‘trast, changing the NaCl concentration had little effect eaither on nH or the apparent Kg for FDP (Figure 16). The \Lariation may be due to a nonspecific ionic strength effect ssince no attempt was made to control ionic strength in these cases. 5. Mn-substrate interactions Results of studying the kinetic interrelationships of 1Nh32+ and ADP or Mn2+ and PEP and the effect of FDP on these iliteractions are given in Figures l7-24. For convenience tdue scales of each set of these two series of figures were iiientically drawn. Generally the following can be said about the results : a. FDP increased the affinity of the enzyme towards Dh12+ at low concentrations of either substrate. 2+ b. FDP reduced nH for Mn at high concentrations of e i ther substrate . 2+ c. FDP reduced nH for PEP at high Mn concentrations. 74 Figure 15. Lineweaver-Burk plot of effect of NaCl on Vm of yeast pyruvate kinase at infinite FDP concentration. Values of Vm including error were estimated from Hofstee plots (Hofstee, 1959) of enzyme velocity and variable FDP concen— tration at constant levels of NaCl. The assays (1.00 ml) contained 100 pmoles Tris—HCl, pH 7.5, 1.0 pmole MnC12, 1.0 pmole triCHA PEP, 1.0 pmole Tris ADP, 33 pg of lactic dehy— drogenase, and 0.15 pmole Na NADH. FDP was added as the tetraCHA salt. Figure 16. Relationship between NaCl concentration and KA of yeast pyruvate kinase for FDP. Values of apparent Kg for FDP were estimated, including error, from Hill plots at constant NaCl concentrations. The plot is derived from Dixon (1953). Assay conditions were identical to those described for Figure 15. 75 - O u - .1 - q — d —1 9 (D l l/Vm (u/mg)" o O) 1 \ J(I I 1 l 1 l 1 1 -4O 0 4O 80 |20 I60 l/S M" (NoCl) Figure 15 1 q l 200 I I keg -1 4.7- 4.5— ] J I I l l l 0.9 I.I 1.3 L5 L? IS 2.: ~LOG [NaCl] M Figure 16 2.3 76 Figure 17. Relationship between Hill slope for Mn and total ADP concentration for yeast pyruvate kinase. Values of n were estimated from Hill plots, varying MnC12 concentration at constant levels of ADP. The assays (1.00 ml) contained 100 pmoles Tris-HCl, pH 7.5, 200 pmoles KCl, 1.0 pmole tri- CHA PEP, 1.0 pmole tetraCHA FDP (when present), 33 pg lactic dehydrogenase, and 0.15 pmole Na NADH. ADP was added as the Tris salt. Figure 18. Relationship between total ADP concentration and apparent KA of yeast pyruvate kinase for MnClZ. Values of apparent KA were estimated from Hill plots, varying MnC12 concentration at constant levels of ADP. Assay conditions were identical to those described for Figure 17. The plot is derived from Dixon (1953). 77 I I I ' l ' I T I ' I ' 4.4- O K‘ with FOP _. . . K+ ._ I" _ N 3.6 _ C: E 1— u 2.8— i -—1 CI 1— Vi\ -1 2.0— K .. 1.2— O _. -e . .43 . J 1 l 1 l 1 J 1 l 1 I 4 2.9 3.3 3.7 4. I 4.5 4.9 . -LOG [ADP] (M) Figure 17 I" NC _I ' I ' I ' I I + I . I I I- 2 0 K wnth FDP V 4.‘ '- + —‘ O K x4 )- - 4.... / .. {.7 1 g—h “—0—. - = \ G) b -1 8 :1 3.3- - O. O __ .. 0 29- 1 ‘53 l 1 I 1 l 1 J, 1 l 1 l L 1 2 .9 3.3 3.7 4. I 4.5 4.9 -LOG [ADP] (M) Figure 18 78 Figure 19. Relationship between Hill slope for ADP and to— tal Mn concentration for yeast pyruvate kinase. Values of nH were estimated from Hill plots, varying ADP concentration at constant levels of MnClz. Assay conditions were identi- cal to those described for Figure 17. Figure 20. Relationship between total Mn concentration and apparent Km of yeast pyruvate kinase for ADP. Values of apparent Km were estimated from Hill plots, varying total ADP concentration at constant levels of MnClz. Assay con- ditions were identical to those described for Figure 17. The plot is derived from Dixon (1953). 79 II I I I I I I1 I I I 4.4? 0K+ with FOP _ - -K" . 3.6- - E - .. 0 SE 2.8- _, ._. I .- CZ P 1”, 2.0- o '3 I .2’" .g. o —I 1 1' I’ 1 I’ 1 10 ?—T 1 1 1 2.9 3.3 3.7 4.I 4.5 4.9 -LOG [Mn2*] (M) Figure 19 8 __ I I I I T I I I I I F _ <1 .".F-'--'r O V 4.I - ‘- E - o——1—o-o“-— .. EC 37... .. E Q) ‘“ _ 5 C1 13£5“- § . '- 8. OK wIth FDP L9 P ”(I q 3 2'9—1 1 l 1 l 1 J 1 l 1 l 1‘ . 2.9 3.3 3.7 4. I 4. 5 4.9 -1_o<3 [Mn2*] (M) Figure 20 80 Figure 21. Relationship between Hill 810pe for Mn and total PEP concentration for yeast pyruvate kinase. Values of nH were estimated from Hill plots, varying MnC12 concentration at constant levels of PEP. The assays (1.00 ml) contained 100 pmoles Tris-HCl, pH 7.5, 200 pmoles KCl, 1.0 pmole Tris ADP, 1.0 pmole tetraCHA FDP (when present), 33 pg lactic de- hydrogenase, and 0.15 pmole Na NADH. PEP was added as the triCHA salt. Figure 22. Relationship between total PEP concentration and apparent KA of yeast pyruvate kinase for MnClz. Values of apparent K: were estimated from Hill plots, varying MnC12 concentration at constant levels of PEP. Assay conditions were identical to those described for Figure 21. The plot is derived from Dixon (1953). I I I T I .1/7' I I I I I 4.41- - )- 0-. ‘ 3.6— _ 3' f- -( 61 C: E 281— .— 3: i .. c t /{ 2.0'“ o o .3 _ c K‘ with FOP. I Jar- 0 0 K7' _- l 1 J, 1 I 1 I 1 I 1 I l 2 .9 3.3 3.7 4. I 4.5 4.9 -LOG [PEP] (M) Figure 21 {.c‘ _I I I I I I I I I I I '- 0 K" with FOP o ' K < )- \ .. x O-no 3.7 I“ § .. 4— 5 .. . x. § 3.3» . _- O - .1 \k}~{ L9 . ... C) ZNSW-l 1 .l 1 J 1 I 1 l 1 Jr 1 -,-' 2.9 3.3 3.7 4.I 4.5 4.9 -LOG [PEP] (M) Figure 22 82 Figure 23. Relationship between Hill slope for PEP and total Mn concentration for yeast pyruvate kinase. Values of nH were estimated from Hill plots, varying PEP concentration at constant levels of MnClz. Assay conditions were identi- cal to those described for Figure 21. Figure 24. Relationship between total Mn concentration and apparent Km of yeast pyruvate kinase for PEP. Values of apparent Km were estimated from Hill plots, varying total PEP concentration at constant levels of MnClz. Assay con- ditions were identical to those described for Figure 21. The plot is derived from Dixon (1953). 83 I I I I I I I I II I I I 4.4 _ 0 K" with FDP. b . K4- _ A 3.6 '- —-1 CL LIJ - .. 9; ..... .1 2.8 - ' g .. I: C. L . ZJDP- o (3 - I .2 — ° -J l 1 I 1 I J l 1 I 4 I 1 2.9 3.3 3.7 4. I 4 .5 4.9 — LOG [Mn2*] (M) Figure 23 a j- I r l I r I I I T I r T‘ 9: 0 K4 with FOP 4. I _- + — E 'K x " I ,_ 3.7 — -4 C: Q) L _ .. 8 N Q 3.3 '- "'I O r- -I 0 2.9 ._ 3 ’1 1 1 1 1 I 2.9 3.3 3.7 4. I 4.5 4.9 Figure 24 -L06 [M‘n2*] (M) 84 d. FDP reduced n for ADP at low Mn2+ concentrations. H 6. Miscellaneous kinetic observations Effect gfglycerol-High concentrations of glycerol (20-50%) protect yeast pyruvate kinase from cold—induced denaturation (Hunsley and Suelter, 1969a). Inclusion of 20% (v/v) glycerol (2.7 M) in kinetic studies identical to those described in Figures 2, 4, and 5 eliminated hetero— 2+ tropic kinetics for K+, PEP, and Mg , mimicking the action of FDP. 2+ Ca activation—Activity with Ca2+ as the sole divalent . . . 2+ cation was about 1% of the rates With optimum Mg concen— trations in the presence of saturating concentrations of FDP and KCl. IV. Binding properties 1. Proton relaxation rate and electron paramag- netic resonance studies . + . . Binary complex-Data for an binding of yeast pyruvate kinase are given in a Scatchard plot (Figure 25). The points lie on a smooth anomalous curve and the enzyme binds up to 6 Mn2+ with apparent affinities which vary by 3 orders of magnitude. The tightest binding site titrated atypically, giving a dissociation constant, KD = 3 FM for the first third of the site and a KD = 72 PM for the remaining two thirds. The 85 Figure 25. Scatchard plot of the binding of manganese to yeast pyruvate kinase. Solutions of the enzyme at 30° were in 50 mM Tris-HCl, pH 7.5, containing 200 mM KCl. The free and bound Mn were determined by the proton relaxation rate of water using a value of 6b = 15.1 obtained by EPR and pro- ton relaxation measurements as outlined in Methods and Mate— rials (Mildvan gt 31., 1970). The curve is arbitrarily fit by four straight line segments. MPK is the titration curve which was obtained for rabbit muscle pyruvate kinase (Mildvan and Cohn, 1965). 86 |.2 I I I O.IOI- ‘ LON- A '7 ‘1’ _ o.oe-' . . I E 0.8- ID .. I.\ ‘ I ' "'- . 9 IVA x =\ ._ 0.6- 004-5“ . "I "a i A a I r? E I L 4: 0v 4 1.... o. I , 0 >- A i I \‘72 1_1 _~ I. 0 I 1 1 1\ akfiqh‘~ C) 2 ‘4 ‘6 0.2” ‘ ~‘~~~~ .1 ~~~.‘ A ~~~~~ ~(MPK V‘V&A~ ~ ~§~~ O 0.5 l .0 I .5 2.0 [Mn]b [YPK] Figure 25 87 next two sites were weaker (KD = 740 PM): and the last 3 sites were weakest (Kb = 1.8 mM) (Mildvan gt a1., 1970). The site-site interaction detected in the Mn binding studies manifested itself only in the affinity but not in the enhancement (Eb) of the bound manganese, which remains at 15.1 t 1.8 (Table 6) as the occupancy of the binding sites increases from O to 6. The correSponding value of 6 b for the rabbit muscle enzyme is 32.7 1 3.2 (Mildvan and Cohn, 1965). Ternary complexes-The addition of all substrates to the yeast pyruvate kinase-Mn complex decreased the enhanced 2+ on the proton relaxation rate of water (Table effect of Mn 6), suggesting the formation of enzyme—Mn—substrate bridge complexes as had been found for the rabbit muscle enzyme (Mildvan and Cohn, 1966). The addition of FDP lowered the é value of the enzyme-Mn complex indicating an alteration of the environment of enzyme bound manganese. Substrate titrations, measuring the relaxation rate of water protons, were carried out at low [Mn]/[EI(O.5 to 0.9) and at high [Mn]/[E](3.S). The resulting values of K3 and E-are given in Table 6 as E(Mn)S and E(Mn)3S complexes, reapectively. Table 6 also gives the apparent Km values and Hill coefficients obtained kinetically with the an+ acti- vated enzyme. 88 o.oav m.H mONH we mom H.mHv o.m moom amm +x o.>Hv 1 1 65m 1 mmmmflcsvm ~.m m.H moom ooa map + +x m.NH o.m moooa Hm +x mmmaczvm o.oHv o.H mama oamav mam H.mHv o.m moo mmm +m 0.5Hv 1 1 oema 1 moamxczvm m.mx o.H mama oom mom + +s o.mH o.~ com com +s aaaxczvm xosma1oav o.oa H.H 000N1OOH manmnum> mom “ooma1ms H.mH m.m1o.m oooa1oo~ magmaum> +x zoomh1vv o o.na 1 1 manmnum> 1 Anson m . a pm ox ms no as macaw mensweax manocnm w Haflm AZLV ucmumcoo coflumfloommfln noum>fluom Xmamfiou m .AOFmH ..mm.mm.cm>paflzv mucmfiwummxm cwumcflx Eoum conga Iuwuwp mum hump EM ucwummmm paw ms .mamflumumz pom mposuwz on ponfiuummp mm musmfifluomxm mums coflumxmamu cououm Eouw pm>nump mum w paw mm pom QM mucmumcou sewumwoommfln .mwumuum 135m pom omwsmmCmE cufl3 mmmcflx wum>5umm ummmw mo mmxmamfioo Mumsumu paw humcflm .0 magma 89 .mam meme :8 A ppm as 21 omH mo mucmmoum aw mam «mufiuu Hmuou mo mEuwu ca pommmnmxmm .mnm mama SE H can a: 21 m0 m0 mucommum an mam muons» Hmuou mo magma c“ pmmmmumxmm .mmm macaw» 28 H can CS 21 oma mo wocmmwum an mam mane Hmuou mo mfiumu an pmmmwumxmm .mmm «Snags 25H ppm :2 21 no mo mocmmoum aw mom mane Hmuou mo mayo» ow pmmmmumxmc .mmm «moan» as H can mom name as H mo mucmmmum :Ho .hufloon> pm>ummno HmmemE mam: pom pmuflsvmu manmflum> wzu mo coflumuucoocoo on» ma Acm acmummmm .Ex ucmummmmv m 0&9 .0 0H H on u e .m.s mm .Hom1mnue :5 om mum3 ucmmmum mucmcomEoo umsuo .pmms mm3 mpfluoacu Esflcofifimamnuofimuumu z oa.o .uoum>«u 10m mo mocmmnm CH .nuon no mam mmomuumu 28 H.H no aux S o~.ouoa.o mum3 muoum>wuum 039m H.mav 1 1 mos +s mum>zu>oxssvm H.mHv 1 1 mm +M macgszvm Apmscflusoov m manna 90 (No significant effect of FDP on the binding behavior of ADP was detected at low or high concentrations of Mn2+. The dissociation constants of ADP measured in the binding studies are significantly greater than those estimated from kinetics. At low concentrations of Mn2+ , FDP weakened the binding of PEP contrary to the behavior of the kinetic data. At high concentrations, FDP tightened the binding of PEP in qualitative agreement with the kinetic observations. Hence E(Mn)3 shows a similar response to the allosteric activator, FDP, in both the binding and kinetic studies while E(Mn) does not. The monovalent activator K+ appeared to raise the affinity of the E(Mn)3 complex for ADP but had no effect on the binding of PEP (Mildvan £5 31.. 1970). 2. Lack of tight FDP binding In order to demonstrate the absence of tightly bound FDP to yeast pyruvate kinase, enzyme was incubated with FDP—14C in the presence of MgC12 (to preserve stability) and chromatographed over Sephadex G—25 (Figure 26). The stoichiometry of FDP binding in the protein peak ranged from 0.01 to 0.1 moles FDP per 165,800 g of enzyme, indicating a 8M). lack of tight FDP binding (KD< ~ 10' In addition, borohydride reduction of a mixture of en- zyme, FDP, and MgCl2 failed to alter the kinetic response of 91 .mamflumumz pom mpocuoz cfl ponfluommp mm mcfiucsoo soflumaaflucflom an pwusmmma hufl>fluumoflpmu ppm coflumuucwocoo samuonm Mom pm>mmmm mum3 mcofluomuw pmuomaaoo mo muosvflam .m.h mm .Homnmflue z oa.o sufl3 mcfluus .xmpmcmmm mmuw Esflpwe um>o muzumummfiwu oEmm mnu um pmcmmnmoumfionno HE om.o ppm 0mm um mmuscfie ma pmumn (SUCH mm3 .m.h mm .Humlmflue mwaofil 00 can AmHOEE\UE mm xufl>fluom Damaommmv U¢H1mom EsflCOEEm DE mnoa x 0.5 .mam mmomuumu mHOEl oo.o .maomz mmaofii 0.6 .wewncm mo mE no.0 mo AmEDHo> Hmuou HE oo.ov muzuxflfi a .mmmcfix oum>su>m ummwm Cu mom mo mcflpcfin mo xomq .mm musmflm 92 (v-v) Ionbuv 1w I'O/UIw/swnoo 0 o 8 § 8 § § “3 IdA Blow/dos selow 60‘) I I I I 4 _. q; _ I / <1 F. -————av—v4"”” -# d dai""—‘—. 4”” _. .777‘-4 '- ~—~——~——--—<~—~.‘--. ° <1 0 \. 1.x. .. \‘.< :1 __ 0!. o Ii, 4——0 4 \A P -1 I I I I I “3 o vommxvmzc mo 0066 cmuammmom oo 1 0602 com Houucoo m «H062 mmmum1uamh ma 0.0H .o.H .mnm amomuumu oom .HmEuoz o momMMIuHmm ma o.H mom omuumu oom .Hmsuoz 0 Hum: ma 6.0H .o.H .mp6 «momhuou oom Hmehoz m ma o.a mom «momuumu oom Hmsuoz a Ammuscflfiv AZEV mcoflufippm Aaom\mlv onDOm 06H» mcflmsoom acofluflppcu H00 wfiwncm mEMNcm H00 .pmuxamumo cH>mHmonflu paw mpflamamuom X m.m mums mama Haa .mmm wusmfim How muo: 00m .mcED (Hoo Hmm mpflEmamuommaom ca 0mmcflx mum>summ ummmm mo mcflmsoom ofluuowamomflouoflz .nmm wusmfim .0&>Nc0 mo cofiumummoum Hmcflmwuo CH popSHocfi mpfluosam Hmsomasmmcmnumfia>cmzmo .mumeSmuwm sufi3 possumfimaomp .m.» mm .H0m1mnhe z oH.o an xmomcmmm m~10 um>o acmxamzv mo 000m cmuammmom mv I 0:02 oomnwwummuu mmzm m.m Q om I 0:02 com HmEuoz Qm.¢ o momuwluamm 06 1 0602 com .Hmehoz m.6 m 00 I mCOZ oom HmEHoz m.o a Ammuscflev AZEV mcofluflppm Aawm\mnv mousom wEflu mcfimsoom Hcofluflppag H00 mfihucm mEhncm opflEmH>uU< x. How .Amm Lonny cmmumouocm 0:» mo mow 0gp um cBocm mum moponumo 0:9 .BOHmn pmuoc mum mcoflu Iflpcoo Hmflommm .mamaumumz paw mpocumz an pannauso mum manpmooum wcu mo maflmumn .pmsmmum Iouosm pcm chum oflumomouoHsufluu ca poxflm cozy .Omm um madmsoow ou pmuommnsm paw conflumfi I>Hom mum3 muouommmm no moumuumnpm ppm .mucwmmmu 0CMEMHMMUM>HOQ .mewusm mo mmamfimm .msEs (Hon How mpfifimamuomaaom cfi 0mmcfix oum>suwm ummw> mo mcflmsoom Uwuuomamomaonoflz .mmm musmwh .ucmfifiuomxm was» ad poms ppm .m.6 mm .H0m1mnhe s OH.o an xmcmnamm m~1w hm>o cmuammmo .m.6 mm .H0m1maua s oa.o an Naoms SE o.oa pom mam «momuumu SE o.H mo manuxfifi m an 0mm um mouscflfi ma pmumnsosamum mm: mfixuch .ucwfiaummxm man» an poms cofluumum =mm nuns: map can .Ahm wusmflmv pomsoommnm maw>aumummmum mm3 wfiwncm m0 me can .ucmEHummxm mwnu aw poms mcofluomum =mm amp: pom 30H: mo 0H5uxwfi m can .Abm wusmwmv pomsoommum mam>wumummmum mm3 wfihucm m0 me can .usmfiwummxm many an poms cofluomum =mm 30H: 0:» ppm .Abm onsmwmv pmmaoommum >H0>flumummmum mmB.0E>N:0 no me can 97 om 1 6:02 com Hopscoo o mcofluomnm mm 30H ma o.H mam 6606606» . 06H .6mmsoommum m «Hows ma 0.0H .o.H .mom «nonhumu com oomumnsocnmum m ma 1 0602 com ocmumnsocnmhm o ucoauomum mm amps ma 1 0602 ooa .6mmsoommum 0 QmGOHuUMHM m0 0H5HXHE ma 1 0602 cos .6mmsoommhm m mc0auomum mm 30H ma 1 0:02 cod .mmhsuommum a AmmuscHEV AZEV mcoflufippm Adom\m1v mouse» menu mowmsoom Hopewuflpp&_ H00 mahucm memucm How .pmuhamumo sfl>mamonwu pom 0pflEmH>uom x_m.o mums mamm Ham .mmm ousmflm Mom duo: 00m .mcfis IHOU Hmm mpflfimawuomaaom ca wannax mum>5u>m unmoh mo msflmnoom owuuomamomflouofiz .umm madman \4.p I vhf-PUZ rufidr‘. fFULu vane-Iv 98 ABCD Figure 28a ABCDE Figure 28b ABCDEFG Figure 28c 99 .m.> mm .Hom1mhue z oH.o 6H xmcmsmmm m~10 um>o acmmavmzv mo 0000 oouHmmmoh oo 1 0coz com Houucou m 00umuuHmm ma 6.0m m Hos com 6 .HmEuoz 6 ma o.OH .o.H Hum: .666 anus oom Hashoz 0 m3 o.H mam mane oom HmEuoz 0 ma o.oH .o.H mHoms .mum>su>0 261mmov oom Hmehoz 0 ma o.H mum>suw0 Zaxmmos com Hmsuoz a Am0ussHEv AEEV mcoHqupm AH0m\m1v 00H500 0EHu mchsoom mcoHqupflH H00 0Ehucm 0E>Ncm H00 .p0umHmumo cH>0HmoQHu ppm 0pHEMH>u00 x m.m 0u03 mH0m HH< .mwm 0nsmHm How 000: 00m .mcE: 1H00 H0m 0pHEmH>uom>Hom cH 0wmcHx 0um>su>m umm0> mo mcH0500m oHuu00H0omHouon .nmm muzmHm oo 1 0602 com Houuaoo a ma 0.0H .o.H «Hum: .004 6006 com Hmeuoz 0 m6 o.H mna mane com Hmauoz n ma 0.0H .o.H «Hum: .606 «mofluu oom Hmsuoz 0 ma o.H 006 among» oom Hmsuoz 0 oo o.OH «Hows oom Hashoz a Am0chHEV AZEV 0coHqupm AH0m\m1v 00u500 06H» mchsoom HcoHqupfiu H00 0E>ucm 0E>Ncm H00 .p0umHmumo cH>0HmoQHu paw 0UHEMH%HUM x.m.m 0u03 0H0m HHa .mmm 0usmHm Mom 0pc: 00m .msED 1H00 H0m 0pHEmH>uothom CH mmmcHx 0um>su>m 0000» m0 mcH0900m oHuuo0H0omHouon .mmm 0usmHm ABCDEF Figure 29a ABCDEI: Figure 29b 101 Opaque gels such as 28a-D were the result of a small amount of protein denaturation. The variation in one gel of the relative position of the two bands to those in another gel resulted from the ionic composition of the gel and had no effect on band intensity or distribution. An attempt was made to separate the two protein hands by slicing an unfixed gel similar to A shown in Figure 28a into 1 mm pieces. Each piece was homogenized in 0.50 ml of 50% (v/v) glycerol—10 mM Na phOSphate, pH 6.5, and assayed by the maximal FDP standard assay. Two activity peaks were found corresponding in position to those made visible by acid fixation. Addition of FDP to the gels (Figure 28b) with or with— out MgCl2 shifted a large portion of the lower fraction (lower pH form) to the upper fraction (high pH form) for both the normal and salt—free enzyme. This conversion was quantitated by scanning gel A from Figure 28a and gel A from Figure 28b at 280 nm in a linear transport device. Distribution of the enzyme in the con— trol gel with no FDP was 55 j 1% high pH form and 45 i 1% low pH form. In the gel containing FDP the high pH form was 81 j 1%.of the total and the low pH form 19 i 1%. The sum of the absorptions from each gel agreed within 8.7%. There— fore, selective destruction of one form cannot account for 102 the change in distribution. (Preparatively prefocused enzyme was refocused in the micro system (Figure 28c). The low pH fraction, high pH fraction, and a mixture of each gave identical distributions on refocusing. The majority of prefocused low pH fraction (F) in the presence of FDP was converted to the high pH form. Enzyme preincubated (D) with FDP (and MgClZ to pro— tect against denaturation), but chromatographed free of FDP just prior to focusing, was skewed toward the high pH form. Similar FDP preincubated enzyme, focused in the presence of FDP and MigCl2 (E), gave a predominantly high pH band. Addition of activators and substrates of yeast pyru— vate kinase other than FDP (Figures 29a and b) had little or no effect on the distribution of high or low pH forms in polyacrylamide gel columns. DISCUSSION The establishment that the yeast pyruvate kinase prep- aration utilized in these studies consists of a single pro— tein species is of fundamental importance for the interpre- tation of the data presented. Disc electrOphoretic experi— ments under a variety of conditions revealed no heterogene- ity, providing enzyme prior to electrophoresis was protected against cold or ligand induced inactivation. The enzyme sedimented in the analytical ultracentrifuge as a single symmetrical peak of extrapolated molecular weight 162,000 to 168,000 in 0.10 M (CH3)4N cacodylate, pH 6.2, containing 100 mM KCl, 26 mM MgCl and 1.0 mM FDP and in 0.10 M Tris— 21 HCl, pH 7.5. Both solvents were similar to those used for kinetic and binding work reported in this thesis (R. Kuczenski and C. H. Suelter, unpublished data). A molecular weight of 165,000 was confirmed with the thantis (1964) high Speed equilibrium technique and strong evidence given for a tetrametic subunit structure (R. Kuczenski and C. H. Suelter, unpublished Observations). Rabbit muscle pyruvate kinase has a molecular weight of 237,000 (Warner, 1958) and consists of 4 subunits (Steinmetz 103 104 and Deal, 1966). Yeast pyruvate kinase, like the rabbit muscle enzyme, has a low extinction coefficient at 280 nm which reflects a low aromatic amino acid complement. Comparison of amino acid mole fractions of the two enzymes reveals, however, marked differences in content (Kayne, 1966), the yeast en— zyme containing about half as many histidine, methionine, and half-cystine residues and about twice as many tyrosine and threonine residues. The value for tyrosine residues per mole enzyme of 38.1 i 1.1 obtained by chemical analysis is more reliable than the value of 47 f 1 obtained by the spectrophotometric method which tends to exaggerate tyro- sine values and to underestimate tryptOphan (Beaven and Holiday, 1952). Thus, the tryptophan content of the yeast enzyme may be higher than estimated here. Partially purified yeast pyruvate kinase has been shown to be completely inhibited by low concentrations of cupric ion (Washio and Mano, 1960). Because of-this observation and because of the low molar ratio of bound c0pper to en— zyme (0.14), an enzymological function of the metal ion is suspect. Efforts to reduce heavy metal contamination low— ered Fe levels to below the limits of detectibility, but were unsuccessful in reducing Cu. The enzyme may simply function, either in vitro or 13 vivo, as an efficient Cu 105 scavenger. The unexpected skewed elution patterns of the enzyme during analytical gel filtration are consistent with the 200,000 molecular weight estimated by Haeckel_g£lgl. (1968) with the same technique. Glycoproteins tend to have an ex- panded structure and chromatograph atypically during gel filtration (Andrews, 1965). However, both,because of the abnormal distribution and the fact that there is no evi- dence that the enzyme contains carbohydrate support the hy- pothesis that the Sephadex matrix induces aggregation of the enzyme (R. Kuczenski and C. H. Suelter, unpublished data). The monovalent cation requirement (Table 5) of the yeast enzyme, like that from rabbit muscle, was both strin— gent and Specific. The substantial activation by Na+ in the presence of FDP may represent a survival mechanism in yeast unessential for muscle, where Na+ only weakly acti- vates pyruvate kinase (Kachmar and Boyer, 1953). Conway and Moore (1954) have described serial bakers' yeast fermenta- tions in the presence of Na citrate and glucose in which 98% of the K+ content of the cell is gradually replaced by Na+. Oxygen consumption of resting cultures of these "sodium" yeasts is about two thirds, and the fermentation rate about one half, of normal. Unfortunately, no data are available on the levels of rate-limiting glycolytic enzymes in these 106 yeasts under these conditions. Wyatt (1964) has hypothe— sized a theory of metabolic control, in which he pointed out that in “systems with several metal activated enzymes and in systems with enzymes with multiple metal activations and inhibitions, many choices of pathways are available." Pertinently, yeast pyruvate kinase was able to utilize Ca2+ as the required divalent cation in the presence of FDP. It, therefore, may not be coincidental that yeast pyruvate ki- nase, whose substrates lie in the reaction sequence of many pathways, has a plastic specificity for activator cations. Kinetically, the yeast pyruvate kinase reaction was 2+-dependent system complex. As studied in the pH 6.2, Mg at limiting K+ or NH4+ but at near saturating concentrations of MgClz and substrates (Figure 3), FDP gave a cooperative activation curve. In addition, FDP acted as a positive heterotropic effector toward K+ and NH4+, Mgz+, or PEP at saturating levels of all other variables (Figures 2, 4, and 5). Since FDP had no effect on the Vmax of the reaction with either K3 or NH4+ as the required monovalent cation (Figure 7), the enzyme may be classified in the nomenclature of Monod gt 31. (1965) as a K system, that is, the presence of FDP modifies only the apparent affinities of the protein 2+ for substrates or metal ions. For the Mg activated sys— tem (Haeckel £5 31., 1968), the appearance of sigmoid ADP 107 kinetics at low concentrations of PEP or K+ were indicative of additional kinetic interactions in the system. Other similar kinetic interactions were apparent for Mn2+ activated kinetics. Lowering of the pH tends to de— 2+ saturation curve in the crease sigmoidicity of the Mn presence of Na+ (Figures 12 and 13). Varying Na+ concen— tration seemed to have little effect on FDP activation for either nH or apparent K: (Figure 16). In the oppoSite case, however, FDP acted as a weak negative heterotrOpic effector toward Na+ and K+ (Figure 9) by decreasing the apparent af- finity. This is in contrast to the action of FDP on KI or NH4+ in the Mg2+-dependent system where FDP acted as a pos— itive heterotropic effector (Figure 2). More importantly, in the presence of K+, strong mutual kinetic dependences of n apparent Km, or apparent KA were HI uncovered between Mn2+ and ADP or PEP (Figures 17—24). FDP 2+ and ADP abolished all kinetic interactions between Mn (Figures 17-20), but Mn2+ and PEP were still able to mutually influence both the affinity or Hill slope of the other (Fig- ures 21-24) under similar conditions. These data do not re- duce to a simple model (Monod gt 31., 1965) partly because in Figures 17-20 resolution of the ADP and Mn2+ variables in quantitative terms of free ADP, free Mn2+, and ADP—Mn com- -4 plex (dissociation constant = 1.0 X 10 M, Mildvan and l.. ._ 108 Cohn, 1966) is dependent on the availability of computer programs to solve polynomials of greater than 3 degrees. For a kinetic system in which n complexes are formed between available ligands (substrates, activators, proteins) and divalent cation, the solution of a polynomial function of (n + 1) degrees is required to determine the free ion con— centration (Kerson gt al., 1967). Therefore, the interac— tions are best discussed as resultant change of enzymatic activity in terms of substrate flux, assuming Mn2+ behaves 2+ Isimilarly to Mg and the enzyme at high dilutions (Srere, 1967) mimics the action in vivo. It is unlikely that the substrates, metal cofactors, or activators of pyruvate kinase.in situ are always at sat- urating levels or at the optimum pH. Consider the effects 2+ of varying the total substrate and Mn concentrations a- round the intermediate level of about 100 pM for each. At this concentration of Mn2+, the only effect of FDP on ADP is to eliminate cooperative kinetics (Figures 19 and 20); for 2+ PEP at 100 pM Mn , FDP greatly reduces both the apparent Km and cooperativity (Figures 23 and 24). Inversely, at both intermediate and high levels of ADP (Figures 17 and 18), the only effect of FDP is to reduce nH for Mn2+. The ap- parent Km for Mn2+ at 100 pM PEP is reduced 5 fold upon ad— dition of FDP and cooperativity decreased but not abolished 109 (Figures 21 and 22). In the glycolytic scheme, after a sudden increase in glucose concentration, the rise in FDP concentration would precede that of the PEP concentration. Applying this tem- poral difference to the kinetic data presented in Figures 17-24 demonstrates that the increase in FDP concentration would first uncouple all ADP—Mn cooperative interactions. Subsequently, the remaining PEP-Mn interactions would be re- leased by the additional increase in PEP, relieving enzy- matic c00perative control (Figures 21-24). The variable stoichiometry of Mn2+ binding in the bi- nary complex (Figure 25) might be explained in 3 ways: a. Aggregation of the protein by Mn. b. Heterogeneity of the yeast pyruvate kinase prep- aration. c. Site-site interaction among Mn binding sites. Aggregation of the enzyme under identical experimental conditions as the binding studies was not detected in the analytical ultracentrifuge. Microheterogeneity was indeed found by isoelectric focusing for this enzyme, which was shown to consist of an approximately equal mixture of two species. Although FDP had a profound effect on the dis— tribution of the bands, no large changes were induced by FDP in the atypical Mn binding curves. The observed 110 conformational heterogeneity does not, then, account for variable stoichiometry of binding. The third alternative of site—site interaction is therefore left. In contrast, the rabbit muscle enzyme binds either 2 or 4 (depending on the monovalent cation present) in either case with an invariant Kb of 63 to 75 pM (Mildvan gt al., 1970). Comparison of the effects of FDP on the binding param— eters of the PEP ternary complexes suggests that Mn2+ must fill at least one binding site before FDP may lower the af- finity for PEP (Table 6). The agreement of the binding data with kinetically determined apparent Km values for PEP, and the lack of agreement of the corresponding parameters for ADP suggest the yeast enzyme-metal complex may bind PEP prior to the binding of ADP, in a preferred order. The results from both preparative and microisoelectric focusing experiments are consistent with the existence in solution of two yeast pyruvate kinase conformers of differ— ing isoelectric point which are resolvable through differ- ences in net charge. It is proposed that the rate for con- version of the forms in the absence of any ligand is slow, but that the allosteric effector, FDP, upon binding the con- former of low isoelectric point, rapidly converts it into the high pH form. Data which support this model are as follows: 111 a. Both bands off preparative focusing columns were kinetically identical in both sensitivity to FDP activation and homotropic response to PEP. b. Variations in distribution of bands in microiso- electric focusing columns were independent of (NH4)ZSO4 concentrations, gel polymerization catalyst, source of en- zyme (whether prepared in the presence or absence of phenyl- methaneSulfonyl fluoride at concentrations sufficient to in— hibit yeast proteases), or addition of any other substrate or cofactor of the pyruvate kinase reaction other than FDP. c. The affinity of the enzyme for FDP was not great enough to form a stable or slowly dissociating complex which might account for net charge differences in the two bands. Besides, binding of this anion would create a complex of lower isoelectric point, not higher, as observed. d. The process is not related to a dissociation-asso- ciation phenomenon. Identical conversion patterns were ob- tained in the presence of FDP or FDP and MgClz. FDP is known to induce dissociation of the enzyme (Kuczenski and Suelter, 1970), which the addition of MgCl2 prevents. The two band pattern is inconsistent with any scheme involving dissociation (or reassociation) of atetramer consisting of identical or nonidentical subunits. e. The conversion process was quantitative. Scans of 112 the gels at 280 nm demonstrate that loss of absorption of the low pH form induced by FDP was countered by a gain of absorption of the high pH form. f. Thekconversion was reversible. Incubation of en- zyme with FDP and MgC12 and gel filtration to remove these ions followed by focusing in gels with or without FDP and MgCl2 showed that a significant fraction had reverted to the low pH form only in the absence of FDP in the duration of the experiment (about one hour). 9. The predominantly low pH form, high form, and a mixture of the two resulting from preparative isoelectric focusing reequilibrated to equal distributions of both forms upon refocusing in microcolumns. The low pH form collected from the preparative column still retained its sensitivity to FDP-induced conversion on refocusing in microcolumns. The microisoelectric focusing experiments provide ad— ditional evidence that the preparation of yeast pyruvate kinase used in this study was molecularly homogeneous by one of the most sensitive separation techniques now known to protein chemists. For instance, the preparation of rabbit muscle pyruvate kinase almost universally used for eXperi— mentation was recently shown to consist of at least 4 sub- species, reminiscent of a mixture of isozymes (Susor et 31., 113 1969) by this same technique. Independent evidence for the existence of two con- formers of this enzyme in solution has been provided by Wiecker _t al.(l969) who found that data from brewers' yeast pyruvate kinase kinetic experiments fitted a simplified al— losteric model (Monod gt al., 1965) which provides for the two conformer state, and Kuczenski and Suelter (1970) whose data explaining the kinetics of the FDP-induced inactivation of the enzyme were consistent with an equilibrium mixture of two or more tetrameric forms in solution. In addition, in— creased activity induced in the basic pH range by FDP may be the result of conformational transitions. There is as of yet no basis for concluding, however, that the hypothesized conformers resulting from kinetic analyses and the double band electrofocusing patterns arise from an identical con— formational mechanism. Further fast reaction kinetic studies may provide clues toward the solution of this problem. Physical separation of "conformers" of a pyruvate ki— nase preparation from rat epididymal fat pads has previously been accomplished, but the conformational change probably involved dissociation and reassociation mediated by a diva— lent cation. In the case of yeast pyruvate kinase, the in- dications are that both conformers remain intact tetramers. SUMMARY The chemical, steady—state kinetic, binding, and con- formational properties of a homogeneous preparation of bak— ers' yeast pyruvate kinase have been studied. The enzyme contained about 0.14 moles of tightly bound Cu2+ per 166,000 g protein and deviated significantly in amino acid content from the rabbit muscle enzyme. Activating monovalent cations were shown to be essential for enzymatic activity. Cooperative kinetics were seen for 2+ K+ and NH +, Mg , and phosPhoenolpyruvate, but not for ADP 4 at saturating concentrations of all other substrates and metal ions, pH 6.2. Fructose diphOSphate heterotropically transformed the cooperative kinetic variables, yielding near hyperbolic saturation curves for each. The enzyme was shown to possess a less stringent specificity for both divalent and monovalent cations in the presence of FDP. Upon addi— tion of PEP or FDP, the activity profile broadened in the range above pH 6.5. For Mn2+-dependent kinetics at pH 7.5, strong interac— 2+ tions were noted between ADP or PEP and Mn , and the effect of FDP on these was investigated. 114 "’1 f 115 Proton relaxation rate and electron paramagnetic res- onance studies of the enzyme binary and ternary complexes revealed an atypical Mn2+ binding curve suggestive of site- site interaction. The qualitative and quantitative agree- ment of the kinetic and binding data support the View that the enzyme—Mn—PEP ternary complexes containing at least one Mn are the kinetically active ones and that there may be a preferred order of binding PEP. 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