ABSTRACT AN ANALYSIS OF THE MECHANISM OF L-RIBULOSE-S-PHOSPHATE 4-EPIMERASE FROM AEROBACTER AEROGENES By Jean D. Deupree L-Ribulose-S-P h—epimerase (EC. 5.1.3.) catalyzes the interconversion of L-ribulose-SAP and D-xylulose-S-P. The mechanism of epimerization was analyzed to determine whether NAD+ was required as a participant in an oxidation- reduction mechanism similar to that of UDP-glucose h-epimerase or whether the mechanism was different. This was prompted by the observation that the L-ribulose-S-P h—epimerase is comparable to UDP-glucose h-epimerase in that: (a) there is no T20 or H2180 exchange with the medium; (b) T is not lost from C-4 carbon of the substrate during epimerization; and (c) the enzyme does not require added NAD+ for activity nor is it inhibited by NADase. However, differences in the mechanisms of the epimerases are implied from the observations that: (a) the kinetic isotope effects differ; (b) L-ribulose-S-P and D-xylulose- SAP are unique among the substrates for the u-epimerases in that a carbonyl group is present two carbons removed from the site of epimerization; and (c) a sensitive catalytic assay did not detect NAD+ bound to the L-ribulose-S-P h-epimerase. Jean D. Deupree Homogeneous L-ribulose-S-P h—epimerase was obtained from Aerobacter aerogenes grown on L-arabinose using the following purification steps: DEAE-cellulose chromatography, ammonium sulfate fractionation. calcium phOSphate gel elu- tion, Sephadex G-200 column chromatography, and DEAE- Sephadex column chromatography. Fine needle-shaped crystals of the h—epimerase were obtained from an ammonium sulfate solution. The homogeneity of the u-epimerase was based on the following observations: (a) the same Specific activity was obtained before and after crystallization and recrystal- lization; (b) a single band was obtained on polyacrylamide gels electrophoresed at 3 different pH values; and (c) a constant molecular weight was obtained across the cell in high-Speed equilibrium ultracentrifugation. A Specific activity of 70 t 10% at 28°C and pH 8.0 was obtained for the homogeneous h-epimerase when Mn?"+ was the only divalent cation present. A molecular weight of 1.14 x 105 t 1.4 x 103 was obtained for the homogeneous enzyme by high-Speed sedimen- tation equilibrium eXperiments. NAD+ was not tightly bound to the h-epimerase as concluded from the following observations: (a) NAD+ was not detected by a microbiological assay either before or after hydrolysis in acid or base; (b) lLAC-NADl' was not incorporated into the u-epimerase isolated from a nicotinic acid-requiring mutant of A. aerogenes which was grown on Jean D. Deupree 1L"C--nicotinic acid and which produced 14C-NAD+; (c) the fluorescence and absorption Spectra of the h-epimerase were not characteristic of bound NAD+ or NADH, and (d) an absorption Spectrum with a maximum around 3&0 mu was not obtained on incubation of the b-epimerase with L-ribulose- 54?. In addition, the homogeneous 4-epimerase did not require NAD+ for activity nor was the activity altered by the presence of NAD+, NADH, NADP+, NADPH in the assay mix- ture. Divalent metal ions activated the U-epimerase to varying extents with the order of activation being Mnf+2> Co++>Ni++> Ca++> Zn++> IvIg++. Incubation of the enzyme with EDTA resulted in a loss of about 90% of the enzyme activity, and the activity was not recovered on passage of the enzyme through a Sephadex G-25 column. However, addi— tion on the divalent metal ions reactivated the enZyme. Indirect evidence is not consistent with lipoate, cystine, BIZ-coenZyme, or an oxidized tryptOphan deriva- tive catalyzing b-epimerization by oxidation-reduction. This is based on the facts that: (a) the absorption Spectra of the u—epimerase were not characteristic of BIZ-coenzyme; and (b) the enzyme was stable to treatment with borohydride, sodium sulfite, or arsenite in the presence of a thio compound,and in the presence of NADH. In additional tests of possible mechanisms, it was concluded that the substrate does not appear to form Jean D. Deupree a Schiff base with the u-epimerase since the enzyme was stable to borohydride reduction in the presence and absence of substrate. Further, a carbanion intermediate could not be detected by incubating the enzyme-substrate complex with tetranitromethane. Thus, it was ascertained that the mechanism of L-ribulose-S-P u-epimerase is different than that of UDP— glucose h-epimerase in that the mechanism of epimerization cannot use NAD+ as an electron acceptor, and the indirect evidence is not consistent with an oxidation-reduction mechanism. A mechanism involving either dealdolization- aldolization or hydration-dehydration is proposed. AN ANALYSIS OF THE MECHANISM OF L-RIBULOSE-S-PHOSPHATE 4-EPIMERASE FROM AEBOBACTER AEROGENES By {x Jean D. Deupree A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1970 3"") ... KS9 '- 31.3” :3 z; This thesis is dedicated to my uncle, Dr. John F. Deupree 11 ACKNOWLEDGMENTS The author extends her sincere appreciation to Professor W. A. Wood for his guidance during the course of this work. The author is further indebted to Dr. Roy H. Hammerstedt for his encouragement, continual interest, stimulating discussions and assistance offered. The author is also indebted to Dr. George N. Holcomb for initiating her into the world of research and for con- tinually encouraging her to obtain a Ph.D. degree. The financial assistance of the National Science Foundation and the Department of Biochemistry at Michigan State University is greatly appreciated. 111 VITA Jean Durley Deupree was born November 9, 1942 in Washington, D.C. She attended Big Rapids High School in Big Rapids, Michigan from 1956 to 1960. She obtained a B.S. in Pharmacy in June 1965 from Ferris State College in Big Rapids, Michigan. She entered Michigan State University in the Department of Biochemistry in June of 1965 where she obtained a Doctor of Philosophy Degree in Biochemistry on May 26, 1970. Jean Deupree is a member of the Lambda Kappa Sigma Fraternity, Rho Chi Society, Sigma Xi and the American Association for the Advancement of Science. iv TABLE OF CONTENTS INT RODUCT ION O O O O O O O O O O O O O O O O O O LITERATIJRE REVIEW I I O O O O C O O O O O O O 0 Chemical Mechanisms of Carbohydrate Epimer- izations O O O O O O I O I O 0 0 O O O 0 0 Enzyme Catalyzed Epimerizations . . . . . . . Aldose 1-Epimerases or Mutarotases . . Epimerizations Adjacent to a Carbonyl I Z'Epimemses o o o o o o e o o o o o B-Epimerases . . . Epimerizations by Oxidation-Reduction UDP-Glucose 4-Epimerase: Studies on the Mechanism . . . . . . UDP-Glucose 4-Epimerase: Other Character- istics . . . Other Nucleoside-DiphoSphate 4~Epimerase IpRibulose-SaPhOSphate 4~E imerase . . . . IpRibulose-S-PhOSphate Epimerase: Studies on the Mechanism . . . . . . L-Ribulose-SAPhOSphate 4-Epimerase: other Characteristics . . . . . . . UDP-Glucuronic Acid 5-Epimerase . . . . . Control of L-Ribulose-SAPhoSphate 4-Epimerase Formation . . . . . . . . . . . . . . . . S Pathway of LqArabinose Metabolism in Bacteria. LnArabinose Operon . . . . . . . . Precedence for a Second L-Ribulose-S- PhOSphate 4-Epimerase . . . . . . . . . quTmIAIS AND PIErHODS C O C C C O O O O 0 O . . . Bacteriological . . . . . . . . . . . . . . . Aerobacter aerogenes . . . . . . . . . . . LeucondStoc mesenteroides LEctoBacIIIus arabionsgs . . . . . . . . . Page 10 10 11 12 13 27 31 31 3L» 35 36 36 37 38 43 43 43 44 44 Chemicals 0 O O O O O O O O O O O O O O O O O 0 General Chemicals . . . . . . . . . . . . . LpRibulose-5aPhosphate . . . . . . . . . . . Enzymat 1c 0 O D O O O O O O O O C O O 0 O O O O L-Ribulose-S-P 4-Epimerase Assay . . . . . . Continuous Assay . . . . . . . . . . . . 2-Step Assay . . Glyceraldehyde-BLP Dehydrogenase Coupled Assay O O O I O O O O I O O O O O O O Phosphoketolase . . . . . . . . . . . . . . Determinations O I O O O O O O O O O O O O O 0 Protein Determinations . . Polyacrylamide Gel Electrophoresis Molecular Weight Determinations . Microbiological Assay for NAD+ . . Identification of1C-NAD+ . . . . RESULTS 0 O O O O O O O O O O O O O O O O O O O 0 Rate of Growth and LqArabinose Utilization by Aerobacter aerogenes . . . . . . . . . . . . Purification of L-Ribulose-S-P 4-Epimerase . Purification Steps . . . . . . . . . . Crude Extract . . . Chromatography on DEAE-Cellulose . Ammonium Sulfate Back Extraction . Ca1cium.PhOSphate Gel Fractionation Chromatography on Sephadex G—200 . Chromatography on DEAE-Sephadex G-50 Crystallization . . . . Other Purification Steps Evaluated . Criteria of Purity of the 4—Epimerase . Other Characteristics of Homogeneous L-Ribulose S-P u-Ep imera 86 o o o o o o o o o o o o o 0 Molecular Weight of the 4~Epimerase . . . . NAD+ Content of the L-Ribulose-SAP 4—Epimerase Microbiological Assay for Nicotinic Acid and NADEAggfigent or’the'ulEoineraée'Arter'cronth -Nicotinic Acid . . . . . . . . . . vi Page 56 6O 62 62 62 63 on 65 65 7O 71 7a 7L» 75 78 81 Page Absorption Spectrum of the 4—Epimerase . . . . 91 Fluorescence Measurements on the 4~Epimerase . 95 Absorption Spectrum of the 4-Epimerase- Substrate Complex . . . . . 96 The Effect of NAD+ on 4-Epimerase Activity . . 96 Other Studies on the Mechanism of Action of L-RlbulOSG-S-P L""‘Eplnlel‘anse o o o o o o o o o o 97 General Discussion . . . . . . . . . . . 97 The Role of Divalent Metal Ions . . . . 113 4-Epimerase Activity in the Presence of Metal Complexing Agents . . . . . . 113 Activity of the 4-Epimerase in the Presence of Specific Divalent Metal Ions . . . . 119 Test for a Carbanion Intermediate . . . . . 129 Effects of Borohydride on Enzyme Activity . . 133 Test for an Electron.Acceptor on the Enzyme Surface 0 O O O O O O O O O O O O O O O I O 136 Other Characteristics of the EnZyme . . . . . . . 138 Anomalous Fast Activity of the Crude Extracts. 138 DISCUSSION . . . . . . . . . . .'. . . . . . . . . . 142 SWMARY O O 0 O O O O O O O O O O O O O 0 O O O O O 16]- RflmmICES O O O O O O O O O O O O O O O O O O O O O 162 APPENDIX 0 O O O O O O O O O O O O O O O O O O O O O 170 vii Table 10 11 12 LIST OF TABLES Typical Purification of L-Ribulose-54P 4-Epimerase from Aerobacter aerogenes . . . NAD+ Content of L-Ribulose-SAP 4-Epimerase Determined by a Microbiological Assay . . . Purification and 14C Content of L-Ribulose- S-P 4-Epimerase After Growth of the u‘n‘ AuxotrOph on Nicotinic Acid (Carboxyi-14C). Identification of 1“C NAD+ by Paper Chroma- 1305373th o o o o o O o o e o o o o e o o 0 Identification of 14C Nicotinamide on Thin Layer Chromatography . . . . . . . . . . . The Effect of Pyridine Nucleotides on the Rate of L-Ribulose-SAP 4-Epimerization . . Divalent Metal Ion Activation of L-Ribulose- SAP 4-Epimerase Based on the 2-Step Assay . Divalent Metal Ion Activation of the 4-Epi- merase Based on the Continuous Assay . . . Divalent Metal Ion Activation of the 4-Epi- merase Based on the 2-Step Assay . . . . . Test for a Carbanion Intermediate Using Tetranitromethane . . . . . . . . . . . . . The Effect of Borohydride on Enzyme Activity . Test for Lipoic Acid and C stine at the Active Site of L-Ribulose-S-P Epimerase . . . . viii Page 61 79 92 94 98 123 125 127 131 135 137 Figure 10 11 12 LIST OF FIGURES Page Mechanism of Lobry de Bruyn—Alberda van Ekenstein Transformations . . . . . . . . 6 Pathways of Pentose, Pentitols and L-Fucose Dissimilation in Aerobacter aerogenes . . 42 The Rate of Growth of A. aerogenes PRL-R3 Versus L-Arabinose UtiIIZation and 4-Epimerase Specific Activity . . . . . . 57 Elution of L-Ribulose-S-P 4—Epimerase from DEAE-Sephadex o o o o o o o o o o o o o o 66 Crystals of L-Ribulose-S-P 4—Epimerase . . . 68 Tracings of Polyacrylamide Gels Containing Crystalline L—Ribulose-54P 4-Epimerase . 72 Molecular Weight Determinations by Sedimen- tation Equilibrium. . . . . . . . . . . . 76 Separatign of L-Ribulose-5-P 4-Epimerase and C on a Column of DEAE-Sephadex . . 84 Separation of 14C and L-Ribulose-S-P a- Epimerase by Electrophoresis on Poly- acrylamide Gel . . . . . . . . . . . . . 86 Dowex-i-Formate Elution Profile of the Perchloric Acid Treated Cells of the u'n‘ mutant which was Grown on 1 C Nicotinic Acid . . . . . . . . . . . . . 89 Proposed Mechanism for Class I Fructose- 1,6-DiphOSphate Aldolase . . . . . . . . 107 The Effect of Metal Chelators on Enzyme AOtI-V1tyo o o o o o o o o o o o o o o o o 116 ix LIST OF ABBREVIATIONS BAL 2,3-Dimercapto-1-propanol DEAE Diethylaminoethyl Ru Ribulose INTRODUCTION L-Ribulose-S-phoSphate 4-epimerase (EC. 5.1.3.)' catalyzes the interconversion of L—ribulose-S-phoSphate and D-xylulose-S-phOSphate. The enzyme is induced in Escherchia coll (1), Lactobacillus plantarum (2) and Bacillus subtiliS (3) by growth on L-arabinose and is induced in Aerobacter aerogenes by growth on L-arabinose (4), L-xylose (5), and L-arabitol (6). Seven different 4-epimerases have been identified thus far. The substrates for all of these, except that for L—ribulose-S-P 4—epimerase, are nucleotide-diphOSphate aldoses which are held in the pyranose form by a glycoside linkage at C-1. In contrast, the open chain substrates, L-ribulose-S-P and D-xylulose-S-P, contain a free carbonyl two carbons removed from C-4 where epimerization occurs. It is currently assumed that the mechanism of all 4—epimerases involves an NAD+-catalyzed oxidation-reduction at C-4 without loss of the migrating hydride ion to the surrounding medium. Evidence for an oxidation-reduction type of epimerization is based solely on studies of UDP- glucose 4-epimerase. Lack of exchange of isotOpic hydrogen and oxygen from labeled water (7, 8, 9) and an inverse isotope effect (10) eliminate an epimerization mechanism involving an Sn2 inversion, carbon-carbon bond cleavage, 1 2 or dehydration. Evidence in support of an oxidation—reduc- tion mechanism include: (a) all UDP-glucose 4-epimerases that have been studied either require added NAD+ for activity or contain tightly bound NAD+; (b) an absorption Spectrum with a 345 mu maximum was obtained upon incubation of the 4hepimerase from.§. 2211 with substrate (11): (c) enzyme-bound NAD+ was reduced with tritiated sodium borohydride, and the T was transferred from the enzyme- bound NADT to TDP-4-keto-6-deoxy glucose (12) to form a mixture of tritiated UDP-glucose and UDP-galactose. Fur- ther, the retention of T on the C-4 position of added UDP- hexose-4—T (13, 14) indicated that the hydride ion which is removed is not free to diffuse from the active site. It is still not known how many steps are involved in the epi- merization of UDP-glucose, or whether hydrogen is trans- ferred only to NAD+ or also to some other component of the enzyme. 0f the other nucleotide-linked 4—epimerases, only UDP-N-acetylglucosamine 4-epimerase (15) has been shown to require NAD+ for activity. There is evidence that some of the UDP-glucose 4-epimerases can catalyze the epimerization of nucleotide-linked aldoses in addition to UDP-glucose and UDP-galactose (i6, 17, 18, 19, 20). L-Ribulose-S-P 4-epimerase is similar to UDP-glucose 18 4-epimerase in that: (a) T or 0 were not exchanged when the epimerization was run in T20 or H2180 (21): (b) T was 3 not lost from C-4 of D—xylulose-S-P-4T during epimeriza- tion:1 (0) the enzyme was not inactivated by treatment with charcoal or NADase, nor did it require NAD+ for activity (4, 2, 21). Although bacterial UDP-glucose 4-epimerases were not inactivated by charcoal or NADase, tightly bound MAD+ was detected in all cases. However, bound NAD+ was not detected on L-ribulose-S-P 4-epimerase from Lactobacillus plantarum using a sensitive catalytic assay (2), The fact that L-ribulose-S-P 4-epimerase was shown to have a different kinetic isotope effect1 than UDP-glucose 4-epimerase, indicates that there are differ- ences in the mechanism of these two epimerases; however, the differences may be only in the rate determining step. Thus, there is a definite need to study the mechanism of 4—epimerases, other than that of UDP-glucose 4-epimerase, in order to substantially determine whether an NAD+-catalyzed oxidationpreduction mechanism is always involved. L-ribulose-S-P 4—epimerase was chosen for this study since: (a) substantial evidence for the presence or absence of an NAD+ requirement for epimerization has not been obtained; (b) epimerization is unknown and may be completely different than that of UDP-glucose 4-epimerase; (c) the substrates are not nucleotide-linked, and thus, it is clear that a nucleo- tide moiety does not participate in the epimerization in 1Fossitt, D., Wood, w. A., Salo, w. L. and Kirkwood, S., unpublished results. 4 any manner; and (d) the enzyme is readily available and has been partially purified from.A, aerogenes. This thesis reports a purification procedure for obtaining crystalline and homogeneous L-ribulose-S-P 4-epimerase from.A. aerogenes. A series of isotOpic, Optical and kinetic eXperimentS Show that MAD+ is not bound to the isolated enzyme and is not involved in the epimerization of L-ribulose-SAP and D-xylulose-S-P. A series of kinetic eXperiments indicate that the epimeriza- tion is activated by Specific metal ions. Further under- standing of the mechanism of epimerization was obtained by determining the effect of inhibitors on enzyme activity. A discussion of the possible mechanism of epimerization is presented. LITERATURE REVIEW Chemical Mechanisms of Carbohydrate Epimerizations Epimerizations of carbohydrates may occur at any carbon by an Snz reaction commonly known as the Walden inversion. The general reaction is as follows: R 18 R R 18 e l‘ H o._| 18 >l‘ e H o: + H>C OH ——> ‘CmOH ——> H o C H + OH I ‘ H'l ‘ I R' R' R' In Snz epimerizations the Opposite epimer is always obtained. Reactions of this type are readily detectable, since, if the reaction is run in H2180, the product will contain one atom of 180 per molecule of product, as shown above. Likewise, if the initial substrate contains 180, the product will not contain 180. Groups on carbon atoms adjacent to a carbonyl group are subject to Lobry de BruynpAlberda van Ekenstein trans- formations (22). The reaction, as shown in Figure 1, is thought to be a general acid, base-catalyzed enolization. To date, these transformations have only been studied to the extent of showing a significant epimerization of an aldose at C-2, considerably more isomerization to the cor- rSSponding 2-keto sugar, and Slight epimerization at C-3. ANNV wcoaumaaommzmae aaopmmoxm cm.» mononaaismsam mp aanoq mo Emasgooz mOIOIm monolm mm ®u m monolm @u m mOIOIm mlolom ONOIm m0! mOIolm monoul OHOIm .II Canada mOIOIm melolm CHOIm 7 A mixture of C-2 and C-3 epimers and the isomerization product is thus obtained. Rearrangement of the carbon chain has not been detectable during the transformations. Metal ions facili- tate the removal of the d-proton from either an aldose or a ketose, probably by forming a complex such as: R I H-C=O. I ';N H-C-o“ I \‘H R! Two of the side reactions of the Lobry de Bruyn- Alberda van Ekenstein transformations are dehydration and aldolization and/or dealdolization. Since dehydration is an irreversible reaction, it will not facilitate the for- mation of additional epimers. Either acid or base aldoli- zations could yield a mixture of four possible epimers. Assuming equal rates of formation of all epimers, the trans, or more thermodynamically stable epimers, would predominate. Aldolization of trioses to form hexoses have been reported in the literature. The conversion of D-fructose to D-Sorbose is thought to occur by dealdoliza- tion followed by aldolization. Although aldolizations are acid or base-catalyzed, they occur during transformations carried out in the presence of a high concentration of free hydroxyl groups and rarely during acid transformations. 8 The mechanism of epimerization at C-1, or mutarota- tion, was first proposed by Lowry (23) as an acid, base- catalyzed reaction which involved the simultaneous trans- fer of a proton from the acid catalyst to the sugar in the same step that a proton was transferred from the sugar to the base catalyst yielding the sugar aldehyde or hydrate directly. ooni‘Blfi:= 0H 0H CI-HHB -> + HA + 13:9 OH There is no indication of carbon-bound oxygen exchanging with the water during the course of the reaction. Others interpret the data as two consecutive bimolecular reactions, where a proton is added in one step and the second proton is removed in a separate step. A mixture of epimers of carbohydrates could also be obtained either by oxidation of the one of the hydroxyls to give the carbonyl followed by reduction or by dehydration followed by rehydration. Enzyme Catalyzed Epimerizations One of the functions of an enZyme is to act as a site-directed catalyst by facilitating the perpetuation of a chemical reaction at a particular site on the substrate and by limiting the amount of the side reactions at other sites on the substrate. Thus the enzyme dictates the 9 epimers which will be formed and limits the amount of side reactions. Therefore, it is not unreasonable to expect most carbohydrate epimerizations to occur by the same mechanism as chemical epimerizations. In fact, many enzyme catalyzed epimerizations are thought to occur by the same mechanism as chemical epimerization although very little supportive data has been obtained. From our cur- rent knowledge, carbohydrate epimerases can be divided into 3-classes based on reaction mechanisms: (a) aldose-1- epimerases or mutarotases, (b) epimerizations adjacent to a carbonyl involving acid-base enolizations (Lobry de Bruyn- Alberda van Ekenstein transformations), and (c) oxidation- reduction mechanisms. Aldose 1-Epimerases or Mutarotases . Epimerization by aldose 1-ep1merases is thought to occur by the same acid, base—catalyzed mechanism discussed for chemical mutarotations. Studies by Bentely and Bhati (24) on the mutarotase from Penicillium notatum indicated that the mechanism did not involve a single diSplacement, dehydration, or a dehydrogenation reaction of any carbon- bound hydrogen or of a hydrated derivative of glucose aldehyde. These conclusions were based on the fact that (a) D and 180 exchange with the solvent was negligible, and the extent of exchange was the same for the enzyme cata- lyzed mutarotations as fOr the Spontaneous mutarotations, and (b) substitution of C-1 hydrogen by D had no effect on 10 the rate of Spontaneous or enzyme catalyzed mutarotation. Epimerizations Adjacent to a Carbonyl Whereas the chemical model predicts exchange of carbon bound hydrogen with protons of the medium, this is not an obligatory process in the enzyme catalyzed processes. For instance in the mechanistically related phoSphogluco- isomerase, Rose (25) found that the rate of incorporation of T into the hexose phOSphate was a function of tempera- ture. At lower temperatures T was not exchanged, and as the temperature increased the ratio of T exchange with solvent to T transfer between adjacent carbons also increased. By conjecture 2-epimerases and 3-epimerases are thought to occur by keto-enolization mechanisms. Only in the case of the 3-epimerase has supportive evidence been obtained. Z-Epimerases: The only 2-epimerases which have been identified are N-acetylglucosamine-6-P 2-epimerase and N-acetylglucosamine 2-epimerase. Ghosh and Roseman (26) have reported a 200 to 300 fold purification of N-acetylglucosamine-6-P Z-epimerase from Aerobacter cloacae. N-glycolyl-D-glucosamine—6-P and N-glycolyl-D-mannosamine- 6-P also served as substrates: however, N-acetyl-D-glucos- amine, N-acetyl-D-mannosamine, D-glucosamine-1-P, D-mannos- amine-l-P, N-acetyl-D-galactosamine-6AP and D-mannose-6-P would not. The activity was not dependent on the addition of any cofactor. 11 Although N-acetylglucosamine—6-P 2-epimerase has not been found in animals, the correSponding N-acetylglucosamine 2-epimerase was found (27). Epimerization of N-glycolyl- glucosamine and N-glycolylmannosamine also occured with the latter enzyme. N-acetylglucosamine-i-P, UDP-N-acetylglucos- amine, N—acetylmannosamine-6-P, N-acetylglucosamine-6-P. glucosamine, mannosamine, glucosamine-6SP, mannosamine-6-P, N-acetylgalactosamine, glucose, and mannose were not active as substrates. The purified enzyme had an absolute require- ment for.ATP, although there was no detectable conversion of ATP to ADP, or AMP. However, Datta and Ghosh (28) obtained evidence that ATP was acting as an allosteric effector with coOperative homotrophic interactions. The mechanism of 2-epimerization has not been studied, although it is thought to occur by the same mechanism as the chemical epimerization of N-acetylglucosamine and N-acetyl- mannosamine at pH 11.0 (29, 30). 3-Epimerases: The existence of a 3—epimerase was first demonstrated by the identification and partial puri- fication of D—xylulose-S-P 3-epimerase from rabbit muscle by Srere 32. El. (31). Since then, the enzyme has been identified from numerous sources. The enzyme from Lactobacillus pentosus does not require a metal ion nor is it inhibited by 10‘2M EDTA (32). Only D-xylulose-5-P and D-ribulose-5-P were found to serve as substrates, although epimerization of tagatose-6-P and xylulose-di-P was tested 12 (33). The enzyme from A, aerogenes did not require MAD+, nor was it inhibited by NADase. McDonough and Wood (21) reported that one atom of tritium was incorporated into an atom of pentose-S-P during the equilibration of D-ribulose- 5-P and D-xylulose-S-P with D-ribulose-S-P 3-epimerase. These results are consistent with a keto-enolization mech- anism: however, the possibility of an entirely different mechanism cannot be ruled out. Anderson and Wood (5) observed the 3-epimerization of L-xylulose-SeP and L-ribulose-SAP in extracts of A. aerogenes. Although the mechanism was not studied, by analogy a keto-enolization reaction is probably involved. Epimerization by Oxidation-Reduction Substantial evidence has been obtained for an oxida- tion-reduction mechanism of UDP-glucose 4—epimerase, and by inferance all other 4-epimerizations have been considered to occur by the same mechanism. It should be pointed out, however, that all but one of the known 4-epimerases cata- lyzes the epimerization of nucleotide-linked aldoses, in which the aldose is fixed in the pyranose ring. In contrast, L-ribulose-S-P 4-epimerase catalyzes the epimerization of the cpen chain substrates, L-ribulose—S-P and D-xylulose-S-P, at a carbon 8 to a carbonyl. Based on our knowledge of chemical epimerizations, it is conceivable.that the mechan- ism of L-ribulose-SAP 4-epimerase involves keto-enolization of the carbonyl and thus may be quite different than nucleotide-diphOSphate aldose 4-epimerases. 13 UDP-Glucose 4-Epimerase; Studies on the Mechanism: A number of mechanisms were originally prOposed to eXplain the epimerization of glucose and galactose. These include: (a) multiple cleavage of the hexose with rearrangement of the carbon atoms to form the epimer, (b) cyclization of the carbon chain to form an inositol ring with cleavage of the ring at a different carbon to form the epimer: (0) attack at C-4 by a hydroxyl group in an Sn2 (Waldenase) reaction with Simultaneous elimination of a hydroxyl group at C-4; (d) dehydration of the substrate with formation of a double bond between C-3 and C-4 or C-4 and C-5 with rehydration to yield the epimer; (e) Splitting of the carbon chain between 0-3 and C-4 with reformation of the chain to give a mixture of epimers: and (f) oxidation at 0-4 with formation of a carbonyl group followed by reduction at C-4 to form the epimer. In 1950, Leloir (34, 35) showed that glucose and galactose were epimerized as nucleotide-linked sugars and not as the free hexoses. If epimerization occurs via inositol ring formation, the UDP moiety would have to be translocated to a different carbon atom. Epimerization by means of multiple bond cleavage of the hexose or by means of carbon atom rearrangement via inositol ring formation has been rather conclusively ruled out by a number of iso- tope studies which Showed that: (a) identical labeling of liver glycogen was obtained from rats fed D20 and a 60% 14 glucose or galactose diet (36); and (b) fasted rats fed glucose-l-luC produced glycogen with 1“C exclusively in the C-1 position of glucose (37). When UDP-glucose 4-epimerase and substrate were incubated in the presence of T20 or H2180, neither T nor 180 were incorporated into either UDP-glucose or UDP-galac- tose (7, 8, 9). If epimerization involves an Snz inversion, 180 should have been incorporated into the sugars. If the mechanism involves dehydration followed by rehydration. some incorporation of T or 180 might be eXpected. However, Rose (38) and Jencks (39) both concur that migration of protons or hydroxyl group from substrate to enzyme can pro— ceed at a rate faster than exchange of the proton or hydroxyl group with the medium. Thus, it is conceivable that the same hydrogen or hydroxyl group which is removed is added back, one of the two being added to the Opposite side of the substrate. The first clue as to the mechanism of action of UDP-glucose 4-ep1merase was discovered by Maxwell (40, 41) when she demonstrated that the purified UDP-glucose 4-epimerase from calf liver required the addition of NAD+ for activity. The following mechanism was thus proposed: 15 NAD+ NADH UDP-Galactose where NAD+ removes a hydride from C-4 of the hexose moiety leaving a 4-keto hexose. A hydride ion from NADH in turn reduces the oxidized intermediate. Further eXperiments indicate that neither NADP+, the d-isomer of NAD+, acetyl-pyridine or pyridine-3-aldehyde analogue of NAD+, nor deamino-NAD+ could be substituted for NAD+. NADase-treated NAD+ was not active, indicating that NAD+, and not a contaminant of NAD+, was the activator. The prOposed oxidized intermediate could not be trapped by running the reaction in the presence of thiosemicarbazide, hydroxylamine or hydrazine. Thus the intermediate must be tightly bound to the enzyme and inaccessible to the surround- ing medium, or a carbonyl intermediate does not exist. The 16 epimerization of UDP-glucose or UDP-galactose was not inhibited by NADH oxidase, acetaldehyde or alcohol dehydro- genase. T was not incorporated into the substrate from exogeneous MAD+-4-T or NADH-4—T which had been added to the reaction mixture. The rate of the reaction in 96% D20 was identical to that in H20. These results indicate that if MAD+ accepts a hydride ion from the substrate, there is a return of the hydride ion to the oxidized intermediate, and the NADH formed during the course of the reaction is not free to exchange with the medium. Since that time, other UDP-glucose 4-epimerases have been found to require the addition of NAD+ to the assay mixture for activity. These include UDP-glucose 4—epimer- ases found in rat tissue (42), human fibroblast lysates (43), tumor cells (44), hemolysates of infants and adults (45, 46), and wheat germ (20). The epimerizations in all cases were inhibited by NADH, and the rate of reaction was dependent on the ratio of NAD+/NADH. In contrast, bacterial UDP-glucose 4-epimerases do not require added NAD+, nor are they inhibited by NADP+ or NADH (7, 9, 48). Tightly bound NAD+ was detected in the epimerase from both yeast (49, 50, 51, 52) and.§. £221 (8) by means of fluorescence in the presence of methyl-ethyl ketone and by enzymatic and fluorometric analysis of the AMD+ released by acetone or perchloric acid denaturation of the enzyme. The yeast and E. coli 4-epimerases Contained 17 1 mole of NAD+ per mole of protein. The enzyme from yeast (9) and Lactobacillus bulgaris (7) were not inhibited by washing with charcoal or incubating with NADaSe. Some of the characteristics of the yeast 4-epimerase have been reported in a series of papers by Maxwell 32, 2;. (9) and Kalckar and associates (50, 51, 52, 53). A fluor- escence emission Spectrum with a maximum at 450 mu was obtained by exciting the enzyme at 350 mu. NADH had similar excitation and emission Spectras. NAD+ bound in the para position to a sulfhydryl group on an enzyme can exhibit characteristic NADH fluorescence as reported for glycer- aldehyde-BAP dehydrogenase (54). The enzyme also contained bound NADH at a ratio of NAD+/NADH between 3 and 10, and the more NADH bound to the enzyme the higher the level of fluor- escence. Thus fluorescence could be due to NAD+, NADH or both. Titration of half of the free sulfhydryl groups with p-hydroxymercuribenzoate resulted in complete loss in enzyme activity. Yet the fluorescence was not completely lost until all of the sulfhydryl groups had been titrated. Bound NAD+ could not be detected in the p-hydroxymercuri- benzoate treated enzyme. The activity, but not the fluores- cence, was restored after precipitating the enzyme with ammonium sulfate and resuSpending the enzyme in the presence of cysteine and NAD+. It should be noted that the liver enzyme (40) was also inhibited by p-hydroxymercuribenzoate, 18 and the activity was restored by incubating with cysteine; yet neither cysteine nor p-hydroxymercuribenzoate had any effect on the activity of the E. 22;; enzyme. Treatment of the enzyme with borohydride in the presence of substrate at the same concentration as the enzyme resulted in a 50-80% enhancement of fluorescence with a concomitant loss in enZyme activity. A similar enhancement of fluorescence was obtained when the enzyme was incubated for 6 to 10 hours with 5' nucleotides in the presence of free sugars. Nucleotides which elicited fluorescence were 5' UMP, CDP, TDP, UTP. Less than a 2-fold increase in fluorescence was obtained with UTP. UDPglucose, UDPgalactose, 5' CMP and 3' UMP. 0f the sugars, D-fucose, D-galactose, and D—xylose produced the same increase in fluorescence which was greater than that obtained with L-arabinose, D-glucose, or D-ribose. Sucrose and L-fucose had a negligible effect on the fluorescence. Uridylic acid and galactose were rapidly incorporated into the protein. The bound galactose was not liberated even after digestion of the epimerase with pronase and trypsin, although it was released on denaturation with alcohol or heat. The bound galactose was converted to a unidentified product even in the absence of 5' UMP. The 4-epimerases which had been either reduced with borohydride or incubated with 5' nucleotides in the presence of sugars were comparable in that: (a) the magnitude and 19 quantum yields of fluorescence were identical; (b) the enzymes had only 5% of their original activity; (c) less than 10% of the original NAD+ was present; (d) only 30% of the increase in 340 mu absorption could be accounted for as NAD-4-H, although the assay used may not have detected all of the NADH. Not enough eXperiments have been carried out at this time to accurately interpret all of these results. The fluorescence enhancement may not be related to any mechan- istic prOperty of the enZyme, and the free nucleotides and sugars may be reacting at a site on the enzyme remote from the active Site. However, it is possible that the 5' UMP and free sugars were reSponding in the same manner as true substrate, in which case, the sugar would have been oxidized at C-4 by transfer of a hydride to NAD+. In the normal reaction the oxidized intermediate would be immediately reduced by NADH, so that at any one time the steady state level of NADH would have been too low to be detectable. However, in the presence of pseudosubstrate, the oxidized intermediate may not have been reduced due to the inability of the protein or substrate to make a necessary conforma- tional change, or the oxidized intermediate may have been converted to a second non-reactive form. Thus, detectable levels of NADH would have been produced, and this would have been observed as an increase in fluorescence. Since the reduction of the enzyme with borohydride was carried 20 out in the presence of substrate, the borohydride may have reduced either NAD+, the oxidized substrate or both, but in either case NADH would have formed which would have pro- duced the increase in fluorescence. Indirect evidence for a reduction of NAD+ during the course of the epimerization was obtained by Wilson and Hogness (11). They found that incubation of the E. 32;; 4—epimerase with substrate produced an absorption Spectrum with maximum at 345 mu. If NAD+ were reduced during the course of the reaction, one would expect an absorption maximum near 340 mu: due to electron perturbation caused by the protein, the exact wavelength will vary from protein to protein. They calculated that 19% of the enzyme bound NAD+ was in the reduced form in the presence of substrate. Their data are consistent with the following sequence of reactions: ....> .__>. NADox-E + S NADox--ES NAD 1, NADox-E + S2 where E is the enzyme and S1 and 82 are UDP-glucose and UDP-galactose. Bevill 33. El' (13) and Kohn.g£.‘gl. (14) indepen— dently presented evidence for retention of T at C-4 during epimerization by the yeast enzyme. Thus, the T is either stereOSpecifically removed from the hexose and reintroduced, or the C-H bond is never broken. 21 To further elucidate the mechanism, Bevill and coworkers (10) studied the kinetic isotOpe effect observed when tritium was substituted for the hydrogen at C-4 of the hexose moiety. A mixture of either UDP-glucose-i-luc and UDP-glucose-4-T or UDP-galactose—i—140 and UDP- galactose-4-T was incubated with the yeast 4-epimerase for given periods of time. The rate of appearance of 140 in the product was taken to be a measure of the rate of reac— tion of hexose with hydrogen at C-4, since any 1“'0 isotOpe effect should have been negligible. The ratio of T/14C at any given point in the reaction to T/14C at equilibrium was plotted versus % attainment of equilibrium. The plots were extrapolated to 0% attainment of equilibrium to obtain an estimate of kT/kH' An inverse isotOpe effect of either 1.5 or 3.0 was obtained depending on the direction of the reaction. If T were transferred in the rate determining step, a positive isotOpe effect would have been SXpected. How- ever, a small or even inverse primary isotOpe effect might have been obtained if hydrogen were bound more tightly in the transition state than in the starting state. Due to the d-hydrogen isotope effect of reactions involving a trigonal carbon, such as cleavage of 0-0 or C-C bonds at C4, the unlabeled compound Should react 12-25% faster than the T-substituted compound. A Sn2 like mechanism could conceivably explain the inverse isotOpe effect; however, 22 180 exchange with the medium would have been necessary for an Snz reaction. An inverse isotope of 3 would be consistent with transfer of hydrogen to oxygen or carbon, but transfer of the hydride ion to sulfur atom is ruled out. Bevill prOposed the following mechanism to eXplain their data: .1; _r. _a3 ___\. E + SH L-Ribulose-S-P 4-Epimerase >»D-xylulose-5-P ———>.Pentose cycle L-Ribulose-5-P 4—epimerase is also induced in E. coli (1), L. plantarum (2), and A. subtilis (3) by growth on L-arab— inose. The pathway of L-arabinose metabolism in all of these bacteria is essentially identical to that in A. aerogenes. IpArabinose Operon Englesberg and his associates (1, 72, 73, 74) have extensively mapped the L-arabinose Operon in E. 222A. The L-arabinose Operon codes for a regulatory protein, an initiator site, L—ribulokinase, L-arabinose isomerase and L-ribulose-S-P 4—epimerase in that order. The L-arabinose permease is located at a different site on the DNA and also contains an initiator site. The regulatory protein, or repressor, presumably is an allosteric protein containing a site for attachment of the regulatory protein to the initiator regions of both the L-arabinose Operon, and the L-arabinose permease gene, and also a site for L-arabinose. 38 Binding of L-arabinose to the repressor is thought to pro- duce an allosteric transition in the repressor to form the activator. The activator initiates the transcription of the genes coding for all of the proteins on the L-arabinose Operon as well as for the L-arabinose permease. A mutation in the initiator gene which results in the production of either more or less of the L-ribulokinase also results in a coordinate increase or decrease in the level of L—arabinose isomerase and L-ribulose—S-P 4-epimerase indicating that synthesis of these three proteins is coordinately controlled. Although the L-arabinose Operon has not been mapped in A, aerogenes, Mortlock and associates have shown that L-arabinose isomerase, L-ribulokinase and L-ribulose-SAP 4-epimerase are coordinately controlled as in E. coli.2 Precedenge for a Second L-Ribulose-5:P 4-Epimerase LqArabitol, L-xylose, and L-xylulose are not natur- ally-occuring pentoses, however, A, aeroggnes is able to metabolize them. The pathway for metabolism of these pentoses was determined by W. A. Wood and associates (6, 76) to be as follows: 2LeBlanc, D., Mortlock, R. P., Deupree, J.. Wood, W. A., unpublished results. 39 L-arabitol ribitol dehydrogenase L-xylulokinase L-xylulose 4“ >‘L-xylulose-5AP L-fucose L-xylulose-S-P isomerase 3-epimerase L-xylose L-ribulose-5-P L-ribulose-S-P 4-epimerase D-xylulose-S-P The questions which remain are: (a) what is the genetic location of the genes coding for all of these enzymes, and (b) is the L-ribulose-S-P 4uepimerase coded for by the gene in the L-arabinose Operon, or is there a second gene. In order to answer these questions it is necessary to discuss the metabolism of pentoses and pentitols, by A. aerogenes in detail. The arabitol dehydrogenase activity present after growth on L-arabitol is the result of a very low rate of oxidation by ribitol dehydrogenase. Further growth of the organism on L-arabitol results in the selection of mutants which are derepressed and constituitive for'ribitol dehydro- genase (77). Likewise, L-fucose isomerase appears to be the enzyme involved in isomerizing L-xylose to L-xylulose, and growth of the organism on L-xylose resulted in the selection of mutants producing constituitive levels of L-fucose isomerase (78). Thus the L-arabitol dehydrogenase 40 and L-xylose isomerase activities appear to be due to pro- teins which are coded for by a gene for ribitol dehydrogen- ase and L-fucose isomerase, reSpectively. The pathway of ribitol and L—xylose metabolism is presented in Figure 2. If L-ribulose-S-P 4-epimerase was translated from the genes of the L-arabinose Operon following growth on these substrates, all of the L-arabinose Operon proteins should have been produced since the L-arabinose Operon is thought to be coordinately controlled. However, extracts of L-arabitol and L-xylose grown cells do not contain L-arabinose isomerase or L-ribulokinase activities (79). In addition, the enzymes of the L-xylulose pathway must not be coded for by the genes in the Learabinose Operon nor are they produced constituitively, since L-xylulokinase and L-ribulose-SAP 3-epimerase activities were not present in extracts of cultures grown on L_arabinose (79). Thus, it seems reasonable to hypothesize that a L-xylulose Operon exists and that it codes for L-xylulokinase, L-xylulose-5-P 3-epimerase and a second L-ribulose-5-P 4-epimerase. This implies that L-xylulose is found in nature and is metabolized by A. aerggeneg. Recently Mortlock2 obtained a mutant of A. aerogenes which was constituitive for the L-arabinose Operon and had a defective gene for L-ribulose-S-P 4-epimerase, in that 4-epimerase activity was not present in extracts of cultures grown on L-arabitol plus casamino acids. However, the crude 41 extracts were able to epimerize L-ribulose-S-P following growth on L-arabitol plus casamino acids. This is highly indicative of the fact that two L-ribulose-5-P 4-epimerases exist, and that each is under separate control and is coded for by separate genes. monomoaom aopompoao< 2H godpmaaaammaa omoosmiq cam mfiopapcom .Owopamm mo madSSpmm .N oaswam AI mnHIOmOH505mIA MI omoaaosmiq A, mmooamnq ommadx ommaomoaoanom minionoaaflmua A onoaafimua A H333 madman Ommaomoaohsoa 2 ommaoaaamim % In». Tl.- oaomo mmOpcom Allll mimiomoasamxlm ommaoaaamiz é, mimnomoasnamuq _AI omoasnamIQ.AlI omoaansa D—Xylulose-5-P Ir—r‘ A504--- Glyceraldehyde-3-P + Acetate TriosephOSphate Glyceraldehyde-3-P g; Dihydroxyacetone- isomerase phOSphate d-Glycerolphosphate dehydrogenase 7 \ > d-GlycerolphOSphate NADH NAD+ The 0.2 ml reaction mixture contained 0.5 umoles of L-ribulose- 5-P, 0.1 umole of thiamine perphOSphate chloride, 1.0 umole NaZHAsou, 0.1 umole NADH, 0.5 umole of MgClZ, 10 umoles of imidazole buffer (pH 7.2) and an excess of d-glycerolphOSphate 49 dehydrogenase, triosephOSphate isomerase and D—xylulose-S-P phOSphoketolase. The reaction was started by adding 4-epi- merase. A molar extinction coefficient of 6.22 x 103 was used to convert the decrease in 340 mu absorbance to umoles of NAD+ formed. This in turn is equivalent to umoles of D-xylulose-S-P formed. Enzyme units are eXpressed as umoles of D-xylulose-S-P formed/min. The continuous assay must be run at pH 7, since phOSphoketolase is not active above this pH. However, it should be noted that the 4-epimerase has a pH Optimum at 9.0 with 40% of the maximal activity at pH 7.0. 2-Step Assay: L-Ribulose-S-P 4-epimerase activity was also determined using a 2-step assay (4), which allowed the L-ribulose-S-P and 4-epimerase to be incubated together in the absence of other enzymes or cofactors. The 0.1 ml reaction mixture, containing 0.5 umoles of L-ribulose-5-P and 5 umoles of glycylglycine buffer (pH 8.0) was preincu- bated for 10 min. at 28°C. The reaction was started by adding 4—epimerase. Aliquots were removed at a given time and added to 2 ul of concentrated acetic acid to stOp the 4-epimerase activity. The enzyme solution was heated in a boiling water bath for 1 min. and then neutralized with NHnOH. The D-xylulose-S-P content, was determined by add- ing an aliquot of the reaction mixture to the remainder of the coupled 4-epimerase assay at 37°C and pH 7.2. The D-xylulose-S-P assay was always started by the addition of phOSphoketolase. The decrease in absorbance due to dilution 50 was determined by running the assay without the addition of 4-epimerase. The D-xylulose-S-P content was determined from the decrease in 340 mu absorbance minus the decrease in absorbance of the assay in the absence of enzyme. The decrease in absorbance was converted to umoles as in the continuous assay. The rate of epimerization followed zero order kinetics up to 6 min. as determined by plotting umoles of D-xylulose-S-P formed versus time. The 2-step assay was used to determine the effect of metal ions on enzyme activity since all metals can be re- moved from the first step of the reaction mixture. To reduce the metal content of the assay to a negligible value, all glassware was soaked overnight in 3N HCl and rinsed with double quartz distilled water. The solutions were not allowed to come in contact with soft glass or metal. The reagents were stored in acid-washed polyethylene bottles. Tris-Hepes buffer was used, since Sigma's Tris has a negligible metal content, and Hepes buffer is a poor metal chelator (89). The pH of the L-ribulose-S-P was adjusted with Tris, and the buffer and the L-ribulose—S—P were passed through a chelex column to remove any contaminating metals. The Tris-Hepes buffer (pH 8.0) was used in the first step of the assay in place of glycylglycine buffer. No attempt was made to remove metals from the reagents used in assaying the amount of D-xylulose—5-P formed or in the continuous assay, Since Mg++ is required by the phOSphoketolase. 51 Glyceraldehyde-3-P Dehydrggenase Coupled Assay: An attempt was also made to assay L-ribulose-S-P 4-epimerase using a coupled assay containing phOSphoketolase, and glyceraldehyde—3-phOSphate dehydrogenase in the presence of AsOu-'-. A slower rate was always obtained using this system. Any contamination with triosephOSphate isomerase resulted in a decrease in the rate of reduction of the NAD+, presumably because of the turnover number of triosephOS- phate isomerase is 100 times faster than that of glycer— aldehyde-3-P dehydrogenase. Thus the glyceraldehyde-BAP coupled assay was not used. PhOSphoketolase D-Xylulose-S-P phOSphoketolase was purified from Leuconostoc mesenteroides using a modification of Hacker's (90) procedure as follows. The pH of the crude extract was adjusted to 4.55 with 1 M acetic acid at 0°C. The precipitate was removed by centrifugation. and the pH of the supernatant was readjusted to 6.0 with phOSphate buffer. The enzyme was applied to a DEAE-cellulose column which had been equilibrated with 0.05 M phOSphate buffer, pH 6.0 and 1 mM thioglycerol until the 280 mu absorption of the eluate was less than 0.04. The phOSphoketolase was eluted using a linear gradient between 0.1 M phOSphate and 0.6 M phOSphate buffer at pH 6.0 containing 1 mM thio- glycerol. The enzyme was precipitated with 3 M ammonium sulfate. The precipitate was back extracted with 2.5 M, 52 2.0 M, and 1.5 M ammonium sulfate in 0.1 M phOSphate buffer, pH 6.0 and 0.01 M mercaptoethanol. Most of the activity was recovered in the 2.0 M fraction and was stored frozen and used as such. Determinations Protein Determinatiggs The protein concentration was determined using the 280/260 ratio as described by Warburg and Christian (91). In preparations containing a very low 280/260 ratio the Lowry (92) procedure for protein determination was used as indicated. The Specific activity is defined as units/mg of Polyacgylamide GelJElgctrophoresis Enzyme fractions were analysed by electrophoresis in 7.5% acrylamide gels according to the method of Davis (93) using square cross-section, Optical quartz tubes (5 x 5 x 100 mm). The lower gel was electrophoresed in Tris-glycine buffer, pH 8.3, to remove the ultraviolet absorbing material prior to applying the enzyme. The enzyme was layered on the lower gel in 5% sucrose and electrophoresed in the same buffer at room temperature. The gels were scanned at 280 mu in a Gilford SpectrOphotometer using a linear tranSport attachment, then removed from the tubes, stained with amido black, and scanned at 560 mu. In some eXperiments, the gels were sliced into 1.7 mm 53 sections using a lateral gel slicer (Canalco, Incorporated). The enzyme was eluted from the gel by incubation in 0.2 ml of 0.05 M phOSphate buffer, pH 7.2, for 4 hours at 4°C. Aliquots were assayed for enzyme activity. The radioactiv- ity was measured by washing the gel suSpension into scin- tillation vials with water and counting in xylene-dioxane- cellosolve scintillation fluid (94) in a scintillation Spectrometer. Molecular Weight Determinations The method of thantis (95) was used for high Speed sedimentation equilibrium studies. The enzyme was sedimented in 12 mm double-sectored cells with sapphire windows in a Spinco Model E analytical ultracentrifuge which was equipped with a regulated temperature control unit and Rayleigh interference Optics. The fringe diSplacement data were analyzed using the computer program of Small and Resnick (96) which had been modified for use on a Control Data 3600 computer. Density of the solvent was determined with a pycnometer. Microbiological Assgy for NAD+ For quantitative determinations of nicotinic acid and NAD+, the micorbiological assay of Snell (97) as described in the Difco Manual (98) was used. The extent of growth of A. arabinose was determined by measuring the turbidity at 660 mu after incubating for 16 to 24 hours at 54 37°C. A standard curve was prepared from the extent of growth obtained with either nicotinic acid or NAD+ run under similar conditions. The enzyme to be assayed was dialyzed overnight against water, and hydrolyzed for 30 min. in a boiling water in the presence of either 0.1 N NaOH or 0.1 N HCl. The samples were sterilized either by passage of 4-epimerase or hydrolysate through a millipore filter or by performing the hydrolysis under sterile con- ditions. As controls, NAD*, glyceraldehyde-3-phoSphate dehydrogenase, NADH, and bovine serum albumin were sub- jected to similar treatment. Identification of 1“C-NAD‘I The 1.”C NAD+ from the u'n' mutant which was grown on 11*C nicotinic acid was partially purified and identified laccording to the procedure of Hagino and coworkers (99). The cells were suSpended immediately after harvesting in a sufficient quantity of 70% perchloric acid to give a final concentration of 5% acid. The precipitate was removed by centrifugation and washed twice with 5% perchloric acid. The supernatant fluid and the washings were combined and adjusted to pH 7 with 5 M KOH. The potassium perchlorate was removed by centrifugation and the supernatant fluid was subjected to chromatography on a Dowex 1-X8 formate (200 to 400 mesh) column (1.5 x 20 cm). The 14 C compound 3 were eluted from the column with 75 mls of H20 and 75 mls each of 0.05, 0.1, 0.25, 1.0, 2.0 and 4.0 M formic acid. 55 The fractions from each radioactive peak were combined and lyOphilized. The residue was taken up in water and lyo- philized two more times to remove the formic acid. The final residue from each radioactive peak was dissolved in water and mixed with cold NAD+ and chromatographed in ascending 1 M ammonium acetate:ethanol (3:7). and 1-butanol:acetone:water (66:1.7z33). and in descending pyridine:water (2:1). In a like manner, the radioactive fractions were mixed with cold nicotinamide and chromato- graphed on thin layer plates using a chloroform:methanol: acetic acid (30:50:20), dioxane:acetic acid (100:1) and chloroform:methanol (90:10) solvents. The radioactive material was detected using a Packard Radiochromatogram scanner: the radioactivity of the paper chromatograms were also detected by audioradioaugraphy. The location of the cold NAD+ and nicotinamide Spots was determined by fluores- cence produced by eXposing to ultraviolet absorption. RESULT83 Rate of Growth And IpArabinose Utilization By Aerobacter Aerogenes A study was made of the growth of A, aerogenes PRL—R3 as a function of L-arabinose utilization and 4-epi- merase Specific activity in order to determine the best time for harvesting the cells so as to obtain the maximum yield of 4-epimerase. A 100-liter culture of A. aerogeges was grown in the fermenter as described in Methods. Samples were withdrawn every hour, and the extent of growth was determined by measuring the turbidity at 600 mu. The cells were removed by centrifugation and frozen, and the supernatant was assayed for L-arabinose content using the orcinol method (86). The frozen cells were subsequently thawed, diluted with two volumes of water, sonnicated, and assayed for 4—epimerase activity and for protein concentra- tion. AS can be Seen from Figure 3, the rate of L-arabinose utilization and the rate of drOp in 4—epimerase Specific activity followed the same curve. The bacteria reached the stationary phase as the L-arabinose content of the medium 3The purification, criteria of purity, molecular weight, all studies on the NAD+ content, and the NAD+ requirement of L-ribulose-S-P 4—epimerase are reported by J. D. Deupree and W. A. Wood in a paper to be published by Journal of Biological Chemistry. 56 57 .Awmv pmop Hoaaoao one maams paopsoo omoaapmamnq pom poammmm mp3 panamaaoaam one .aoHpmapaooaoo zaopoaa pad apaaapom ommaoEHQOIS pow Cezanne was godpmwSMHapcmo an pmboaoa macs mHHoo 0:9 .npzoaw mo Chances a no Spawn no: Spam pzwaa Bo H a mean: 15 owe pm godpaaomnm 0:9 .oopmoaoza mead» exp pm nachospaz one: mpodvdad .mUOSpoz SH ponaaomop mm moapapamsv ampaa ooa ma azoaw mm: mmlqmm homomoaom .¢ aaaaaaoe oaaaooam onoawaaamne can aoaaduaaapa omoaapmagtq mamao> mmlqmm moaowoaom .¢ mo szoao mo opmm 029 .m medmam 58 AIIAIIOV ogigoeds OSDJOUJId3-17 8 <0 apom onHooam “w: omv endaoaaaOIS mlmiomOHSpHmiq addendaadwm Soapmpaoadpom an godpmcaaaopoa pswao3 amazooaoz .m oaswam 77 See or 00.00 N000 00.00 V0.00 00.00 ®NO0 N_.O0 mmmv 1 \m o\ \ 0.0.. 0.0: ”N: 0._- uogiouuaouoo 10 0| 78 and absorption Spectra. To determine if the reaction required added NAD+, homogeneous enzyme was assayed in the presence and absence of added NAD+. Microbiologigal Assayflfor Nicotinic Acid and NAD+ Lactobagillus arabinosus requires nicotinic acid for growth, although it can use NAD+ or NADH as a source of nicotinic acid. Therefore, if L-ribulose-S-P 4-epi- merase contains NAD+, A. arabinosus should be able to use the enzyme or its hydrolysate as a source of NAD+. To test this possibility homogeneous L-ribulose- 5-P 4-epimerase (Specific activity, 13.0) was subjected to a microbiological assay for nicotinic acid using the con- ditions described under Materials and Methods (Table 2). With or without hydrolysis of the L-ribulose-S-P 4-epimerase the bacterial growth was always only slightly above back- ground (Table 2). The enzyme contained less than 0.1 mole of NAD+ per mole of 4-epimerase based upon a molecular weight of 1.4 x 105. One mole of NAD+ per mole of 4-epi- merase Should have been readily detectable, since between 1.7 x 10'”3 and 3.5 x 10'“3 umoles of enzyme were used, and the lower detection limit of the assay is 4.0 x 10",4 umoles of NAD+. To insure that the growth of A. arabinosus was not blocked by the presence of treated epimerase, NAD+ was added to some of the assays as an internal standard prior to the hydrolysis. Under these conditions, the bacterial growth was only slightly higher than that Obtained with 79 m.m Sioa M :.m 0202 ommamwoaoanoo o oposamosalm opmnooHSHOOAHo 0a a m.a mica a m.m mocz z a.o AnoaoSs m.av oez + nan mmmaoaaaOId meimmoaandmtq DNIOH N 0.: muoa N m.m Hum z H.o Amoaoam 0.5v aflz + ommaoaHQOId mumaomOHSDHmnq Nuoa w m.e muoa w m.m modz z a.o cnmaoaaaots mtmuomoaspamuq mnoa a m.: muoa w m.m Hum z a.o onoaoaaaous mumuonoaanamuq mica N m.m enioa N o.m Ocoz OnwaoaaaOi: mtmuowoasnamnq :Hopoam no oaoalwm macaw g paopaoo +Q~ 5.1;: / 2‘6 . — m<\ L_"/,.gfl| I "Q- ~ , \J ,. xmeH: no Momma case on» scam madamom .pswaa poaoabmapas on wSHmoaxm no poosooam mosoowoaosaw esp an cocaaamump was +042 mnp mo soapoooa on» Ugo .Hmficmom BonOPQBOHSooapwm pamxomm w wSHms popompmp was Hwaaopda mbapomoapmh one .Aaumv hmpmznocdpahma wmapmoommp ma pad .Ammun.anoov hmpmzuocopoomuaozmpsnla Uzm Amumv Honmnpm «opmpmom anacoaao 2 H wzficcoomm 2H poaonOpmaoano pzo+aapooncmp Honda on» mo madamoh UmuaHHSQOAH 0:9 mQQonOmeOHSo Hoamm an +Q ‘7" 2 3 N/ l N/ t N H H as proposed by Schellenberg (111). Since NAD+ is known to catalyze the oxidation of UDP-glucose and UDP-galactose in the presence of UDP- glucose 4-epimerase, it can be reasoned that any constitu- ent on the enzyme surface with an oxidation potential similar to that of NAD+ should also accept electrons from the substrate. Lipoic acid (E; = -0.29) and cystine (E; = -0.33) have reduction potentials comparable to that of NAD+ (E; = —o.32). Therefore, it is conceivable that cystine and lipoic acid could act as an electron acceptor and donor in the oxidation-reduction of L-ribulose-S-P and D—xylulose-SAP in the presence of L-ribulose-S-P h-epimer- ase, assuming the reduction potentials for the pentulose phOSphates are similar to that of UDP-glucose and UDP- galactose. If the L-ribulose-S-P 4-epimerase mechanism consisted of an Snz (Walden) inversion at C-#, the hydroxyl at 0-4 would have to be diSplaced by a hydroxyl group from water. However, Wood and McDonough (21) have reported that equi- libration of L-ribulose-S-P and D-xylulose-S-P in the presence of H2180 and h—epimerase resulted in incorporation 104 of less than 13% of the 180 SXpected. This result indicates that a mechanism other than Snz inversion must be involved. Chemical epimerization by an aldolization-dealdoli- zation mechanism have been reported as discussed in the Literature Review. Enzymatically catalyzed aldolizations have been studied extensively as indicated in recent reviews by Morse and Horecker (113) and by Rutter (114, 115). Aldolases facilitate the base catalyzed aldolization between a ketone and an aldehyde as follows: ? R R R _ n I l $20 B: BH C=O C=0 BH B:- C=O ' ' 2:4 ' Hz-C-X M H-C-X H-C-X H-C-X .‘e I - I _ H-C-O H-C-OH 6+ 5 I I H-C=0 R' R' I R. The enzyme catalyzes the exchange of a proton between the carbanion and water. The carbanion is stabilized by dis- persing the negative charge into an electron Sink at the active site of the aldolase either by means of a metal ion or by reasonance of a schiff base with the enamine form. The metaloaldolases (Class II) are found in bacteria and blue green algae (114, 115). All the enzymes in this class require metal ions for activity, and in most cases the metal is tightly bound Zn++, but loosely bound Fe++ or Co++ have been reported for fructose-1,6-diph0Sphate aldolase 105 from Clostridium and Anacystig. The reaction is completely inhibited by metal chelators, and the inhibition is reversed by the addition of divalent cations. The activity is markedly stimulated by K+ and NH“? at a concentration of 10‘1M. The substrate does not appear to form a Schiff base with the enzyme Since the reaction is not inhibited by sodium borohydride. Rutter (11“) has prOposed the follow- ing mechanism for fructose-diphOSphate aldolase which utilizes the metal as an electron sink as follows: 0 0 0 H I _ I _ HZ-C-O-H-O' B:' BH H2-0-0-s-0 Hz-C-O-P-O :3: g; I H-C-OH g 2529 H-C-OH . . "" 0.. ' M o o o Enz I 'M...Enz H-c-O”’ HO-C-H + M++ x I I H-0:0’ H H I R In contrast Class I aldolases do not require metal ions for activity, nor are they inhibited by metal chelators (113). The carbonyl group forms a Schiff base with lysine as demonstrated by reducing the ketimine Schiff base with 106 NaBTu and identifying the tritium labeled intermediate. In addition, the carbonyl oxygen is exchanged for 180 of H2 180 The prOposed mechanism (113) for Class I fructose-1,6-diphos- phate aldolase is presented in Figure 11. Based on the mechanism proposed for fructose-diphos- phate aldolase, the following mechanism can be proposed for L-ribulose-S-P huepimerase: HZ-c-OH C=0 + HZN-Lys I HO-C-H I HO-C-H I I? Hz-C-O-P-O‘ O Hz-C-OH l H 0:N+—Lys B:- -0H' I _a£; +OH' I Ho-c-H 9 Hz-C-o-H:0 0 Hz-C-EH 0:N+—Lys I H0 C-H <é—-——€> .9 0=C-H I I’ HZ-C-O-P-O' I 0' HZ-c-gH C=N-Lys I +0H' I “-OH' H-C-OH I 0' Hz-C-OH I I1 C=N+-LyS l HO-C-H l ‘O-C-H I 9 _ HZ-C-O-E-O 0‘ HZ-C-QH 0:N+-Lys I HO-C-H I H-C-O’ I 9 HZ-C-O-P-O' O HZ-f-OH 0:0 Ho-c-H H-|C-0H HZ-C-O-H-O' O 107 AMHHV ommaoca< mumsamonoamI0.anmouoshm H mmoao how amacmzooz vomOQOMm .aH opzmam sash ceasapox one aHem csaaapox 0o ocoaosoofls -e I. -o -s .: IOIanucumm IOImuououwm Ionwuououmm IOIHIOIUINm IOImIOIUINm a. _ i - . _ . _ . _ o o o o monoum monIm mOIoIm omononm mOIUIm . . _ _ mOIoIm mououm mouoIm Iououm onoum _ _ . . muunos muouom muouom muouom . ommu _ +m+ _ _ m I IIIIIV I IIIIIIV . I . I 653.72 m + ouo AINIII 23-26 Alli £3326 j 23-126 0 + I . Io _ m .0 +m Io m _ u I0 m . . N n . N - m mm . IouanIoI m Ionauououmm -oumuouon m noumuououmm 2 I ._ . o o o o omom each ceasapcx aaaaom censuses monmvm mOIoINm moIoumm mouoINm . _ um: . o~m+ N _ 2.7+. no 3 mmqutauo Allllllll/ mSIzuo 4|IIII|.MIIIV 23:2 m + can 0 m +m+ o m- .0 _ mm Ium .0 I0 .0 Ionmuonoumm Ioumuouonmm -oumuoIOINm IoumIoIUINm o o o o 108 The enzyme could also act as an electron sink by forming a Schiff base between the substrate and enzyme or by means of a metal ion. The 2- and 3-carbon intermediates would not have to be released from the enzyme surface, and thus there would not be necessary for any proton exchange at C-3 as found for transaldolase where dihydroxyacetone remains tightly bound to the enzyme surface and protons are not exchanged with the medium (113). The h-epimerase would have to prevent the racemization of the hydroxyl at C-3 and permit the racemization of the hydroxyl group at C-4, with the trans or more thermodynamically stable isomer predominating. In contrast, aldolases catalyze either a Egggg- or a gi§-, but not both, arrangement of the hydroxyl groups on carbon-carbon bond formation. This appears to be a prOperty which is dictated by the fructose-diphosphate aldolase since the condensation of pyruvate and glyceralde- hyde-3-P by 2-keto-3-deoxy-6-P gluconate aldolase always gives 2-keto-3-deoxy-6AP gluconate and not 2—keto-3-deoxy- 6-P galactonate (116). Dehydrations of hydration reactions between carbons aand B to a carbonyl are catalyzed by a number of different enzymes including fumarase, aconitase, enolase, and nucleo- tide diphOSphate aldose reductase. There are at least 4- classes of hydrases and dehydrases (117) including: (a) enzymes requiring cofactors, (b) enzymes requiring both a metal ion and a reducing agent, (0) enzymes requiring a 109 divalent metal ion, and (d) enzymes requiring pyridoxal phosphate. Of the two basic mechanisms, only the one of which fumarase and enolase are characteristic is applicable to L-ribulose-S-P u-epimerase. In both of these enzymes, two-imidazole groups, one charged and one uncharged, are believed to be present in the active site and are believed to act as proton donors and acceptors as follows: :»r~““”'N “‘\\ /,- ~a~w~ N . I / H I H H ‘ f H \ 07 . I I XHHIIRIVCI: X /C\ I 6 ‘x / ' \ C Race-Y -——_-— \I' R'_C RooooY \ / H/ OH H I HO H \ H N \\\::: N This mechanism requires a polarization in the substrate which could be induced by a dipole in the catalyst, so that the proton donor and acceptor could also be reSpon- sible for this polarization of the substrate. However, in the case of imidazole groups, where the proton acceptor is uncharged, this does not seem possible. Instead, the polarization is thought to be induced via the interaction of the substrate binding groups, x and y, which would include cofactors such as Mg++ in enolase. The free energy changes associated with hydration-dehydration reactions are, in general, small so that these processes have equilibrium contents close to unity. The overall reaction catalyzed by 110 aconitase is similar to that expected for L-ribulose-S-P u-epimerase in that with aconitase a hydroxyl group is translocated to an adjacent carbon, whereas, with L-ribulose- 5-P 4-epimerase the hydroxyl group would be translocated to the Opposite Side of carbon atom. The prOposed aconitase mechanism is (118): H H COOH H COOH I \ / \ / (D)H-C-COOH C C I -0H‘ ,“ -H(D)+ I HO-C-COOH --——4> (D)H _____;> I I +OH- ‘. +H(D)+ H-C-COOH ‘C C I / \ / H CH COOH CH. COOH 2 | 2 COOH COOH -OH‘ +OH’ H I HO-f—COOH (D)H-C-COOH I H-C-COOH I H which proceeds by both a trans addition and a trans elimin- + ation of water. Aconitase requires both cysteine and Fe+ In comparison to known hydration and dehydration reactions, the following mechanism can be prOposed for L-ribulose-5AP h-epimerase: HZ-C-OH 0:0 I HO-C-H I HO-f-H 9 HZ-c-o-H-O' CD HZ-f-OH Bz‘ BH- 0:0 ' I Al) HO-C:- I HO-C-H I 9 _ Hz-C-o-H-o 0“ HZ-C-OH GD I 0:0 I +OH' HO-C-H _____;> I ‘< ““.T' +0-H “OH I 9 _ Hz-c—o-H-o 0' 0 H2-0—0H I 0:0 ~OH' I .._.___..; HO_C HOH' I C-H H H 0 E 0' 2’ ' ',: 0 Hz-F-OH 0:0 I HO-C-H I H.lc-0H9 H2"C"O-ri -0- 0- Slight variations in the above mechanism could exist, such as steps 1 and 3 could be acid catalyzed and involve keto- enolization HZ'f“OH C=O liq, HO-C-H I HO-C-H I ?_ HZ-C-O-H-O 0’ +H -H+ as follows: H2-?-OH +H+ I 0:: ITO-C) -H+ I HO-C-H I 9 Hz-C-O-P-O‘ o- HZ-C-OH I 0:”6N ;;::::ii Ho-C +H20 " C-H I O H -C-O-H-O 2 I 0' dz-f-OH C=O I HO-C-H I 4. I” 0 ll _ HZ-C-O-P-O I- O 112 A metal could act as an electron Sink at the carbonyl group and as a Lewis acid to accept the hydroxyl group at C-4 as follows: H\ [H H\ /H H\C/H ,/ ‘0H ,/ 0H // ‘0H (B\C (;3—fi ,0:0 \§,H /(“\O \ /OH MZH M 0 CC/ 0H QM KHC/IC/ <——-—> \OH II} 0 H’C \\ H H C\. H 2 H/ \\ /H 0’ 0 0\ // ‘H / H // H \ ,, 0\ ,0 0\ 90 I /P\ /..P\ - 0- \0' 0- 0‘ 0 0 H H H H \C/ \C: // ‘0H // OH _ 0:0 0:0 BH B. .‘ \\ ,H \\ [H .' 0 c\ i Z x / ‘OH H\ / 0H M H—c+ M /0 \:,2 H H0 \\ ,H 3 0H 0: 0\ H // H 0\ 90 0\ 40 P P 0‘ ‘0‘ 0‘ \0' A base is required to accept the proton from the d-carbon Since the d-proton does not appear to be excahngeable with the medium (21). The metal ion could facilitate the trans- location of the hydroxyl to the Opposite side of C-4, or the carbonium ion could rotate. In either case there should be an equal probability of the hydroxyl group returning to either side of C-4, and the more thermodynamically stable 113 epimer should predominate. The above reaction is not freely reversible since the equilibrium of step 2 is far to the right. However, starting with either L-ribulose—S-P or D-xylulose-S-P a mixture of L-ribulose-S-P and D-xylulose- 54F would be formed from the preceeding mechanism. A better understanding of the mechanism of L-ribulose- S-P h-epimerase could be obtained if it was established whether (a) metal was involved in the enzyme reaction, (b) the substrate formed a Schiff base with the enzyme, (c) a carbanion was formed during the course of the reac- tion and (d) an electron acceptor other than NAD+ was present on the enzyme surface. The following eXperiments were undertaken to answer these questions. The Role of Divalent Metal Ions h-Epimerase Activity_in the Presence of Metal Com- plexing Agents: L-Bibulose-S-P h-epimerase did not require the addition of divalent metal ions for activity at any stage of the purification; however, the apprOpriate pre- cautions were never taken to remove divalent metal ions from the enzyme of the assay mixture. A more definitive indication of whether an enzyme requires a metal ion for activity can be obtained by assaying the enzyme in the presence of metal chelators. Reactivation by addition of divalent metal ions would be indicative of a metal require- ment by the enzyme. Therefore, the u—epimerase activity was determined in the presence of a number of metal chelators. 114 The continuous coupled assay could not be used to determine the activity of the b-epimerase in the presence of metal chelator, since Ng++ is required by the phoSpho- ketolase, one of the coupling enZymes. Therefore, the enzyme was assayed in 2—steps. Step 1 consisted of incu- bating the u—epimerase with L-ribulose-S-P in the presence of glycylglycine buffer. The reaction was stopped by the addition of acid, and the enzyme was irreversibly denatured by heating in a boiling water bath for one minute as described in Methods. Step 2 consisted of assaying for the amount of D-xylulose-S-P which was formed in step 1. This was accomplished by adding an aliquot of the reaction mix- ture from step 1 to the coupled assay containing phOSpho- ketolase, triosephOSphate isomerase, and d-glycerolphos— phate dehydrogenase as described in Methods. The D-xylulose- S-P concentration was calculated from the amount of NADH oxidized during the course of the reaction. To determine the effect of metal chelators on enzyme activity, h-epimerase (50% pure), which had been dialyzed overnight against 0.05 M glycylglycine buffer, pH 8.0, was incubated at room temperature in the same buffer with 1 mM o-phenanthroline, 0.1 M dithioerythritol, 0.1 or 1 N mercaptoethanol. 2 x 10‘3M and 8 x io-ZM 8-hydroxyquinoline sulfonate, 10-3M BAL and 10-3 or 10'2N EDTA. Aliquots were withdrawn at given time intervals and assayed for h-epimerase activity. Precautions were not taken to remove the divalent 115 cations already present in the assay buffer or the glass- ware used in either the first or second step of the assay. The addition of metal chelator to the second step of the assay did not interfer with the assay for D-xylulose-S-P when added at a concentration comparable to that carried over from the first step of the assay. However, o-phenan- throline at levels higher than 1 mM did interfere with the D-xylulose-S-P assay. A plot of the percent of activity remaining after given periods of time is illustrated in Figure 12. with- out metal chelator the h-epimerase lost 20% of its activity in the first 20 min. and remained stable thereafter. Only EDTA and 0.08 M 8-hydroxyquinoline sulfonate inactivated the enzyme within 20 min. The enzyme was initially acti- vated by 1 M mercaptoethanol. dithioerythritol, and o-phenan- throline, and even after 3 hours the level of activity remaining was still higher than that of enzyme incubated in the absence of metal chelators. The extent of activa- tion was always less than 2-fold. BAL and 2 x lo-3M 8- hydroxyquinoline sulfonate had essentially no effect on enzyme activity. The effects of metal chelators on the u—epimerase activity may be due to their ability to remove divalent cations or else they may be due to a nonSpecific reaction of the enzyme with the chelator. Based on the list of stability constants for metal chelator complexes given in 116 Figure 12. The Effect of Metal Chelators on Enzyme Activity The 4-epimerase preparation approximately 50% pure, was dialyzed against 0.05 M glycylglycine buffer, pH 8.0 and incubated with metal chelator in 0.05 M glycylglycine, pH 8.0, at room temperature. An aliquot was withdrawn at the times indicated and assayed for u—epimerase activity in the 2-step assay at 37°C and pH 8.0 as described in Methods. All values are eXpressed as percentage of the activity remaining compared to the activity prior to the addition of the chelator. 117 I75 g....f..’.3‘\ k.— I M Mercaptoethonol If ‘5 ISO 0 .E l25: .E O 8% a: IOO . .t.’ \ sulfonate -E IO’3 M BAL \_,\\ o 75 \. 4 \ q ------ ~~~~- \ \ ”‘wo‘ / %5 ' Io”M :Y 50 \ Mercaptoethanol ‘3“ \0 ° -2 ‘ 0 \€\ 8 x lo M - B-Hydroxyquinoline \ sulfonate A n) 01 / .\‘._.‘/. ‘\ IO'3 M EDTA 4O 80 IZO Time (min) B-Hydroxyquinoline ISO ‘2 200 118 the Appendix, BAL, glycylglycine, and 8-hydroxyquinoline sulfonate, and o-phenanthroline bind Zn++, and Ni++ better than Mg++ and Ca++, and EDTA binds all metals tightly. Thus EDTA is probably complexing all the divalent cations present in the enzyme solution, whereas the other chelators should be complexing Specific divalent cations and leaving other metal ions still free in solution. Hence, the results presented in Figure 12 could be eXplained if the u-epimerase were able to bind all metal ions in solution but was activated to different extents by the different metal ions, with Ca++ being more active than Zn++. Thus, the purified enzyme may consist of enzyme molecules with various activities which are dependent on the metal ion which is bound. Chelators, such as BAL, may remove the metals from the medium which have lower abilities to acti- vate the h—epimerase and, thus, allow the binding of divalent metal ions which produce higher activities. Inactivation produced by metal chelators only after a lag of 3 or more hours was probably a non-Specific inac- tivation. The metal chelators should have rapidly bound the cations in solution. The rate of complexing of metal ions bound to the enzyme surface would depend on the rate of release of the metal ion from enzyme surface. However, most metal-containing enzymes are inactivated in less than 3 hours. 119 Activity_of the h-Epimerase in the Presence of §pecific Divalent Metal Ions: In order to determine if EDTA was inhibiting the enzyme by removal of the divalent cations or by some other means, it was necessary to show that the enzyme could be reactivated by metal ions after treatment with EDTA. Thus, if EDTA were inactivating the enzyme by some other means, the activity should be regained on removal of the EDTA. If EDTA were inactivat- ing the enZyme by complexing a necessary metal ions, the enzyme activity should be regained only after addition of the metal ion. To test this, it was necessary to remove all metal ions from the solutions and the glassware as described in Methods. A Sephadex G-25 column (0.6 x 11 cm) was exten- sively washed with 10‘2M EDTA to remove metal ions and was then extensively washed with metal free 0.05 Tris-Hepes buffer, pH 8.0. Following incubation with 10'2M EDTA for 1 hour, there was no detectable epimerase activity in the 2-step assay, but a Specific activity of 2.2 was obtained with the continuous assay containing Mg++ compared to a Specific activity of 3 by both assays prior to the EDTA inhibition. The enzyme inactivated by EDTA was passed through a Sephadex G-25 column which had previously been shown to clearly separate 32F and blue dextran. In order to insure separation of EDTA and h-epimerase only the fractions 120 containing most of the enzyme were collected. After passage through the Sephadex column the enzyme had no detectable activity in the Z—step assay but had a Specific activity of 4.2 in the continuous assay where Ng++ was present. This experiment was repeated 5 times, and the activity in the 2-step assay was recovered after the Sephadex step only when metal ions were present. In preliminary eXperiments, when the enzyme was incubated at room temperature with a series of metal chloride salts at 10'3M concentration and assayed in the 2-step assay at 28°C, activities comparable to those presented in Table 7 were obtained. Of the divalent metals studied only Cu++ and Fe++ were found to interfere with the second step of the assay when added directly to an assay of a known amount of D-xylulose-S-P. In addition the other divalent cations did not cause the chemical epimerization of L-ribulose-SéP as determined by adding the cation to the first step of the assay in the absence of u—epimerase. The results of the 2-step assay indicated that more than 90% of the enzyme activity was lost on incubating the enzyme with EDTA. Although EDTA in solution should have been separated from the h-epimerase on passage through a Sephadex G_25 column, tightly bound EDTA may not have been removed. The enzyme activity lost on EDTA treatment was recovered on the addition of the divalent metal ions indi- cating that the enzyme requires metal ions for activity. 121 In addition, as much as a 17-fold activation over the activity after dialysis was obtained depending on the metal ion added. The order of activating effect of the metal ion was Mn'H' > C6”) N 1'”) Ca++> Zn++> Mg'H'. The enzyme in the presence of 10'3M Mg++ regained less than 40% of the original activity. It should also be noted, that when the enzyme was incubated overnight at u°c with io-2H EDTA, only about 50% of the protein eXpected was recovered from the Sephadex G-25 column, indicating that the enzyme had probably been denatured or adsorbed. Metal containing proteins are often denatured when completely depleted of metal ions, although the EDTA may inactivate the enzyme in other ways over the extended period of time. In order to show more conclusively the order of activation of the divalent cations, the 4—epimerase after recovery from the Sephadex G-25 column was assayed in the presence of varying quantities of cations. The metal salts used were freshly prepared solutions of Spectrographically analyzed metal sulfates containing less than 5 parts per million of most other metals. Since the same activity was obtained when the enzyme was preincubated with 10'3H Co“'+ for O, 10 or 30 min. the enzyme was not preincubated with metal ion prior to assaying. Rather metal ion and substrate were preincubated to allow temperature equilibration of the assay mixture, and the reaction was started by the addition of enzyme. 122 The results presented in Table 7 indicated that the highest h—epimerase activity at the lowest divalent cation concentration was obtained with Mn++. A 17-fold stimula- tion over starting activity or a 70-fold stimulation over activity of the enzyme in the absence of any metal ion was obtained with Mn++ at 10'5M, whereas higher concentrations were required for the maximal activation possible by Ni++ and Mg++ These results also substantiated the hypothesis that EDTA inhibited the enzyme by removing necessary cations and not by binding to the enzyme. If EDTA were still bound to the enzyme after its recovery from the Sephadex G-25 column, and if the inhibition of the enzyme were due to the presence of the EDTA, the initial activity should have been recovered with Zn‘"+ and Ni++ at 10"LL M or at a lower concentration than would be obtained with Mn++, since the stability constant for an"+ and Ni++ EDTA complexes are ion-fold higher than that of Mn++. However, enzyme activ- ity was obtained with Mn++ at a 10-fold lower concentration than with Zn++. In additional experiments the enzyme after dialysis and after recovery from the Sephadex G—25 column was diluted in 10-4H C080” and 10-3M MnSOu, ZnSOu, MgSOu and N180“ and assayed in the continuous assay. Neither the assay mixture nor the glassware had been treated to remove metal contaminants and a syringe with a metal needle was 123 Table 7. Divalent Metal Ion Activation of I—Bibulose-S-P h-Epimerase Based on the 2-Step Assay The 4-epimerase (85% pure) was dialyzed overnight against 0.05 M Tris-Hepes buffer, pH 8.0, incubated for one hour with 10-2M EDTA and passed through a Sephadex c-25 column (0.6 x 11 cm) which had been washed free of cations with EDTA and equilibrated with 0.05 M Tris-Hepes buffer, pH 8.0. The 2-step assay mixture contained SpectrOpure metals at the levels indicated in the Table. Precautions were taken to remove the contaminating metals from the glassware and the reagents as described in Methods. Metal Ion Concentration (M) Specific Activity Conditions None 10-6 10-5 10'“ 10-3 After dialysis 3.u After dialysis and EDTA 0.85 Mnso,+ 51 57 59 60 C0304 32 37 50 60 NiSOu 2,2 4.3 21 is Ca012a 8.0 9.6 ZnSOu 0.93 3.9 3.3 3.4 MgSOu 0.38 0.29 0.29 1.3 aCaCl2 was Malinckrodt analytical reagent grade. 124 used to measure the enzyme aliquots. The results of the continuous assay, as presented in Table 8, do not reflect the activation of the 4—epimerase by Mn++, Zn++, or Mg++ that was obtained with the 2-step assay (Table 7). The difference in activities obtained by the two assays is most likely due to the presence of contaminating metal ions in the continuous assay. However, results of the continuous assay do indicate that Co++ and Ni++ can acti- vate the metal free 4-epimerase, although the activity as determined by the continuous assay was only 50% of that obtainable by the 2-step assay. The exceptionally low activity obtained with Hn++ is probably due to the fact that phOSphoketolase is inhibited by high concentrations of Mn?"+ (119). Likewise any of the metals may be inhibit— ing the coupling enzyme in the assay, and thus the reac- tion may not be zero order. Hence, it is not possible to clearly interpret the results of the continuous assay, and the only conclusion which can be drawn is that an accurate measure of enzyme activity in the presence of Specific divalent metal ions cannot be obtained with the continuous assay. It should also be noted that enZyme which has not been freed of contaminating metal, cannot be activated more than 2-fold by any metal as determined by the 2-step assay. For this experiment the 4-epimerase (90% pure) was dialyzed for 2 hours against 0.05 M barbital buffer, pH 8.0, 125 Table 8. Divalent Metal Ion Activation of u-Epimerase Based on the Continuous Assay The enzyme was treated as described in Table 7. After dialysis and EDTA additions, and after elution from the Sephadex column, the enzyme was diluted in 10-“M 0080 and 10'3M Mn++, Zn++, Mg++, and.NiSOu (SpectrOpure) and assayed in the continuous assay as described in Methods. Specific Activity Ratio of: Conditions After Dialysis After EDTA and Continuous Assaya Elution from Sephadex G—25 2-Step Assayb None 3.27 “.06 1.2 MnSOu 2.23 5.9 0.099 C0804 2.93 22.1 0.43 N1304 3.52 8.1 0.h5 ZnSOu 1.86 2.22 0.65 MgSOu 2.53 3.08 2.4 aThe Specific activity obtained after EDTA treatment and passage through a Sephadex G-25 column. bThe Specific actiVlty obtained from the 2-step assay (Table 7). 126 incubated for 1 hour with 10'2M Co++. Hn++, Zn++, and Hg++ chloride salts, and assayed in the 2-step assay. Contamin- ating metals were not removed from any of the glassware or reagents. The results presented in Table 9 indicate that only Mn++ can stimulate the enzyme in the presence of "endogeneous" metal ions. However, only a 2-fold stimula- tion was obtained indicating that Mn++ was not able to completely overcome inhibition by the other cations, since a 17-fold activation is obtained in the absence of any com- peting metal ions. Neither Co“'+ nor Mg++ markedly changed the activity, but it is not possible to determine from this experiment whether Co++ or Mg++ became bound to the enzyme. These results also substantiated the 2-fold stim- ulation observed with some of the metal chelators. The observation, that Co++ appeared to activate the enzyme 5-fold as determined by the continuous assay and not at all in the 2-Step assay, can be eXplained by the fact that a metal complexing buffer was used in the contin- uous assay and not in the 2-Step assay. Imidazole binds heavy metals readily and prevents inactivation by these metals, whereas barbital is a very poor metal chelator and thus does not remove any of the competing metals ions (see Appendix). Since the preceeding results strongly indicated that L-ribulose-E-P h-epimerase was activated by metal ions, Mn++ being the most active, it was necessary to 127 Table 9. Divalent Metal Ion Activation of the u-Epimerase Based on the 2—Step Assay The L-ribulose-S-P h-epimerase (Specific activity 10.0) was dialyzed for 2 hours against 0.05 M barbital buffer pH 8.0 and incubated for 1 hour with 10-2h Co++, Mn++, Zn++, and MgC12 and assayed in the 2—step assay in the presence of glycylglycine buffer pH 8.0 and any metals already present in the buffer, substrate or glassware. Conditions % of Original Activity C0012 89 MnCl2 205 ZnCl2 l3 128 redetermine the Specific activity of homogeneous u-epimer- ase in the presence of Mn*+. Therefore h-epimerase which was 80% pure as determined by acrylamide gel electrOphoresis, was crystallized twice as previously described. The second crystals were washed with 1.8 M ammonium sulfate, collected by centrifugation and resuSpended in 0.05 M Tris-Hepes buffer, pH 8.0. The 2nd crystals were at least 98% pure as determined by polyacrylamide gel electrophor- esis. The enzyme was treated with 10"2 M EDTA, and the EDTA was removed by passing through a Sephadex G-25 column as before. The metal-free enzyme was incubated with 10-“M MnSOu (Spectropure) and assayed with the 2-step assay to which 10'5M MnSOn had been added. A Specific activity of 70 i 10% was obtained for the pure L-ribulose- S-P H—epimerase. EDTA at a concentration of 10‘3M completely inhibited the enzyme in the presence of 10-4M Mn++, however, 10'3M 8-hydroxyquinoline sulfonate did not inhibit the enzyme when this concentration of Mn‘"+ was present. After the excess Mn++ had been removed by passage through a Sephadex 0-25 column, incubation with 10-3M 8-hydroxyquinoline sulfonate resulted in a 50% inhibition, whereas incubation at a concentration of 10'2M resulted in complete loss of enzyme activity. These results indicated that the enzyme can be inactivated by chelators other than EDTA. The differences in levels of the two chelators required to 129 inhibit the enzyme can be eXplained by the fact that the binding constants of EDTA for Mn++ is 10‘3 times higher than that of 8-hydroxyquinoline sulfonate. In order to determine if Mg*+ could inhibit the 4-epimerase in the presence of Mn++, the enzyme was pre- incubated with either Mn++ or Mg++ and assayed immediately in the presence of the Opposite metal. Only 10% of the activity was lost when the enzyme in the presence of 10'uM Mn‘"+ was diluted in 10'3M Mg++ and assayed immedi- ately in the presence of 10'3M Mg++, however 50% of the activity was lost when the enzyme was assayed one hour after diluting. Likewise, when the enzyme in the presence of 10'3M Mg‘++ was diluted and assayed in 10-“H Mn++, the enzyme was only 50% as active as it was in the absence of Mn*+. Thus Mn++ is capable of inhibiting the enzyme in the presence of Mn**. Testffor a_Carbanion Intermediate Tetranitromethane reacts with carbanions with the liberation of nitroform which absorbs at 350 mu. Riordan (120, 121) has reported that tetranitromethane reacts with the enzyme-substrate complex of both yeast (Class II) and muscle (Class I) fructose diphOSphate aldolases. The theory that tetranitromethane is reacting with the car- banion intermediate of the aldolase reaction is based on the following observations: (a) the rate of production of nitroform from tetranitromethane is markedly enhanced 130 by the presence of substrate, (b) the rate of nitroform production is directly proportional to the concentration of active enzyme, (c) the substrate concentration result- ing in half-maximal rate of nitroform production (Km') was virtually identical with the values of Km of fructose 1.6-diphOSphate and fructose-i-phoSphate determined enzy— matically, (d) phoSphate competitively inhibits the tetra- nitromethane reaction, and (e) the substrate is consumed as a function of time. Thus it is reasonable to assume that if 4-epimerization of L-ribulose-S-P and D-xylulose- S-P is proceeding via a carbanion intermediate; it may be possible to detect the carbanion by conducting the epimer- ization in the presence of tetranitromethane. To test this the 4—epimerase was dialyzed for 5 hours against 3-changes of 0.05 M glycylglycine buffer, pH 8.0. The 0.2 m1 reaction mixture contained 0.05 M buffer, pH 8.0, 4.2 x 10'“M tetranitromethane, L-ribulose- S-P, and h—epimerase. The initial change in absorbance at 350 mu was used as a measure of the rate of the tetra— nitromethane reaction. As illustrated in Table 10, both the substrate and the h-epimerase (80% pure) reacted with tetranitromethane when incubated separately with tetranitromethane in all three buffers. However, pure h-epimerase did not react with tetranitromethane in Tris buffer. Tetranitromethane did not appear to react to the 131 Amasav owmaoadaoudn Amaze Rwy mammoaaaminm 000.0 000.0 000.0 000.0 00.00 0N.a mass 000.0 000.0 000.0 000.0 b0.00 0m.a wane 000.0 000.0 000.0 000.0 n0.0~ 00.0 maaa 000.0 000.0 000.0 000.0 00.0 00.0 mass 000.0 0aa.0 000.0 000.0 00.0 0N.H osaoaawaaoaao 000.0 00a.0 ~a0.0 000.0 00.0 0m.a ceaoaawaaoaao 000.0 000.0 000.0 H00.0 om.0a 0.0 odouooaaH 000.0 000.0 000.0 000.0 00.0 00.0 oHowooaaH 000.0- 00a.0 aa0.0 000.0 00.0 00.0 caowwoaaH EM!“ 2 mo." K S aoaesm + ommhoafiamue magnum + Mohmsm + cocchoMMaQ + mnmusmlq omdhoaanmu: minusmiq composaamlé mimiomoasndmlu magnum asaaxas 00040 opom HoapasH .mopafiouosmohpooam onouado 0 ca comm pd SSH soapomoh on» Mon pendanopou was :8 0mm pm oosmnnomno SH owedno HdapaSa one .oopoodSSa ms ommnoaaaoi: one .opdhmeSm .deSpoa uoupasoupop SSIOH N N.: .o.m ma .hommsn z no.0 confidence bananas Soapodon Ha m.o one msmspoEOHpHCSHpoB wade: onwaooapoch sodsmnamo d How pmos .oa canoe 132 enzyme-substrate complex, since the rate of the tetranitro- methane reaction with L-ribulose-S-P in the presence of huepimerase was not Significantly greater than the combined rate of the tetranitromethane reaction with substrate alone and enzyme alone. In addition increasing the amount of enzyme did not increase the rate of the tetranitromethane reaction with enzyme-substrate complex. However, using the same conditions, it was possible to obtain a net initial increase in 350 mu absorbance for muscle fructose— diphOSphOphate aldolase when tetranitromethane was incubated with the enzyme fructose-diphoSphate complex. The rate of the tetranitromethane reaction was dependent on the aldolase concentration. The reaction of the substrate with tetranitromethane may be due to the ready removal of the d-proton on the sub- strate at basic conditions. This would result in a carbanion formation which could be stabilized by keto- enolization of the carbonyl group. Also the substrate may form a Schiff base with the glycylglycine thus facil- itating the removal of the d-proton. The reaction of tetranitromethane with u-epimerase (80% pure) was probably due to the reaction of tetranitro- methane with the contaminating protein. Tetranitromethane is known to react with both tyrosine and sulfhydryl groups on proteins (122). Under the conditions used a carbanion intermediate 133 of the L-ribulose-S-P haepimerase complex was not avail- able to react with tetranitromethane in quantities which could be detected. Thus either a carbanion intermediate is not formed during the course of the epimerization, it is too Short lived to react with tetranitromethane, or tetranitromethane is not freely accessible to the Site of carbanion formation. Effects of Borohydride on Enzyme Activity If the enzymatic h-epimerization of L-ribulose-S-P and D-xylulose-S-P proceeds by either a dealdolization- aldolization or dehydration-hydration mechanism, the reaction would be facilitated by acceptance of the elec- trons from the carbonyl group of the substrate by a group on the enzyme, as previously discussed. In Class I aldolases this is accomplished by formation of a Schiff base between the substrate and the lysine residue of the enzyme (112). The Schiff base is readily reducible in all Class I aldolases. Therefore, it can be reasoned that if a Schiff base is formed between either L-ribulose- S-P or D-xylulose-S-P and the enzyme, the enzyme-substrate complex Should be reduced by sodium borohydride rendering the enzyme inactive. However, the enzyme should not be inactivated on incubation with borohydride in the absence of substrate. If the mechanism of epimerization involves an oxidation-reduction mechanism a substituent on the enzyme 134 such as lipoate, cystine, Biz-coenzyme or oxidized trypto- phan would have to act as an electron acceptor or donor as previously discussed. Lipoate (123), cystine, and the indolenine group of oxidized tryptOphan (110) are reducible by borohydride. Therefore, if the mechanism involves oxidation-reduction, it may be possible to inhibit the enzyme by borohydride reduction of the electron acceptor; however, the substrate should protect against the boro- hydride reduction. The borohydride reduction was performed using the procedure reported by Inghram and Wood (124). To 1.6 x 10’“ umoles of L-ribulose-S-P 4-epimerase (85% pure) in 0.2 ml of 0.05 M phOSphate buffer (pH 7.0) was added 2 ml of 1 M NaBHu followed three min. later by 2 ul of 1 M acetic acid. The NaBHu and acetate additions were repeated three times after whichinialiquot of the enzyme was removed and assayed for enzyme activity. Three more additions of NaBHu and acetate were made and the enzyme was reassayed. Likewise, the reaction was run in the presence of 2.5 x 10'3M L-ribulose-S-P and additional substrate was added after the first of the three additions of NaBHu. The reaction was also run in the presence of enzyme L-ribulose— 5-P and 10'”M CoClz. As Shown in Table 11, only when the enzyme was incubated with NaBHu was as much as 30% of the activity lost. Part of the loss can be attributed to loss in activity of the enzyme on standing in buffer during the 135 Table 11. The Effect of Borohydride on EnZyme Activity The 0.2 ml reaction mixture contained 1.6 x 10'“ umoles of L-ribulose-S-P huepimerase (85% pure) and 10 umoles of phoSphate buffer, pH 7.0. To this was added three additions of 2 ul each of NaBH followed three minutes later by 2 ml each of 1 M acetic a id. An aliquot was removed and assayed for enzyme activity, after which the additions were made again. The same reactions were al 0 run in the presence of 2.5 x 10'3M L-ribulose-S-P and 10-EM CoClg as indicated. L-Ribulose-S-P was added a second time at the beginning of the second borohydride treatment. -— Additions Activity Remaining (%) 1st Borohydride 2nd Borohydride Treatment Treatment None 90 70 + L-Ribulose-S-P 96 80 + L-Ribulose-S-P and CoCl2 98 136 first hour. The other reactions were run later and had a lower u-epimerase activity prior to the addition of sub- strate. Likewise the u-epimerase at pH 6.5 in 0.05 M phos- phate buffer was not inactivated by borohydride either in the presence or absence of substrate, however, MAD+ was reduced under these conditions. These results strongly indicate that a Schiff base is not being formed during the course of the reaction; however, one cannot rule out the possibility that a Schiff base may be forming at a site on the enzyme which is not accessible to borohydride. The results also support the theory that the enzyme does not contain bound MAD+, since the enzyme should have been inactivated by borohydride in the absence of substrate. Both lipoate and cystine are readily oxidized by 02, and thus if they were reduced during the course of the reaction, they may have been reoxidized prior to assaying; hence, inactive enZyme would not have been detected. Test for an Electron_Acceptor on the Enzyme Surface The epimerization of L-ribulose-S-P and D-xylulose- S-P may proceed by an oxidation-reduction mechanism using enzyme bound lipoic acid or cystine as an electron acceptor, as previously discussed. If this were true, then the h—epimerase would probably be inhibited by either arsenite or sulfite, since dihydrolipoate and cysteine 137 irreversible react with arsenite, and lipoate and cystine irreversibly react with sulfite. To test this L-ribulose-S-P 4-epimerase (85% pure) was incubated with 10’1H, 10'2M, io-3M and 10-“h sodium sulfite as described in Table 12 and assayed for enzyme activity in the continuous assay. In Similar manner, the h-epimerase was incubated with either 10'2M mercaptoethanol or 10-3M dithiotheritol to reduce the disulfide bond. The "reduced" enzyme was then incubated with sodium arsenite and assayed for enzyme activity in the continuous assay. AS illustrated in Table 12, L-ribulose-S-P h—epi- merase was not inactivated by sulfite or by arsenite in the presence of either mercaptoethanol or dithiotheritol. If either lipoic acid or cystine was present in the active site, the enzyme should have been inactivated by either of these treatments. However, the enzyme may have pro- tected the lipoic acid or cystine from reacting with the arsenite or the sulfite. Other Characteristics of the Enzyme Anomalous Fast Activity of the Crude Extracts Two levels of n-epimerase activity, which differed by a factor of 3 to 5, were found in the crude extracts. The higher level of h-epimerase activity was always lost on the first purification step, and only the lower level of h-epimerase activity was recovered. Although this 138 Table 12. Test for Lipoic Acid and Cystine at the Active Site of L-Ribulose-S-P h-Epimerase The 0.h>umoles of L-ribulose-S-P h-epimerase (85% pure) was inc bated at room temperature with 10'1M, 10' M, 10'3M and 10- Na 30 in 0.05 M Tris-Hepes buffer pH 8.0. The activity remaigin after 30 min. was determined using the continuous assay as described in Methods. Alternatively the enzyme was incubated as above with 10‘2M mercaptoethanol or 10‘3M dithiotheritol. After 30 min. sodium arsenite was added to a concentration of 10‘1M or 10-“M. Aliquots of the enzyme were removed after 5 min. and assayed in the continuous assay. Additions Relative Activity Zone 100 10-“H Na2803 98 io-3H Na2803 100 10'2M Nazso3 90 10-1M Na2803 98 io-3M NaAso2 90 10‘1M NaA802 96 10'3M Dithiotheritol 100 10'3M Dithiotheritol + 10-“M NaAso2 95 io-3M Dithiotheritol + 10-3h NaAso2 100 10'2M Mercaptoethanol 100 10‘2M Mercaptoethanol + 10-“M NaA502 95 10'2M Mercaptoethanol + 10'3M NaASOZ 95 10'2M Mercaptoethanol + 10'2M NaA802 9o 10'2M Mercaptoethanol + 10'1M NaAsOZ 90 139 phenomenon was not conclusively explained, a number of studies were conducted to help clarify this situation. The anomalous activity of the crude extracts con- sisted of an initial fast rate of NADH oxidation which tapered off within 5 min. to a slower linear rate. The fast rates were not reproducible and did not follow zero order kinetics. However, an accurate determination of the 4-epimerase Specific activity is difficult to obtain for the crude extracts due to NADH oxidase activity as dis- cussed previously. To denature the NADH oxidase, the crude extracts were heated to 60°C at pH 7.0 or 8.0 in either Tris or phOSphate buffer. Although there may have been some loss in 4~epimerase activity, the anomalous fast activity was still present in the heat treated extracts. If an aliquot of the extract was preincubated with the reaction mixture for 10 min. prior to the addition of L-ribulose-S-P, the initial fast rate was no longer observed. All of the components of the reaction mixture were necessary to obtain the fast activity. Additional experiments indicated that the anomalous fast activity was lost if the enzyme solution was allowed to remain at 4°C for 3 to 4 days, but it was not lost dur- ing storage at -20°C. The fast activity was also lost on dialysis, ammonium sulfate fractionation or on DEAE cellu- lose chromatography. The total enzyme activity recovered from the DEAE-cellulose step was comparable to that calcu- 140 lated from the slow linear rate observed with the crude extracts, but this was three to five times lower than that calculated from the initial fast rate. There was no enhancement in activity when either boiled crude extract, filtrate from enzyme concentrated by ultrafiltra- tion, or when concentrated dialysis buffer was added to an enzyme assay. The high level of activity found in the extracts from the u'i' mutants does not appear to be related to the high level of activity found in extracts of the wild type, since the high level of enzyme activity found in extracts of the mutant was not lost on purification, and the level of pure enzyme obtained from the mutant was approximately three times higher than that obtained from wild type. The anomalous fast activity found in some of the crude extracts may be comparable to the actual activity of the enzyme in the intact cell. On rupturing the cell the protein may irreversibly change conformations to a less active form. The rate at which the protein changes con- formation may vary from one cell preparation to another depending on the harshness of the treatment used in ruptur- ing the cell wall. On the other hand, the anomalous fast activity may be related to the ability of divalent metal ions to acti- vate the purified enzyme. One would eXpect most divalent metal ions to be present in the crude extracts. However, 141 the concentration of metal ions will depend on the extent of binding of the metal ions to all proteins present. Thus the activity of the 4-epimerase Should be a reflection of the different divalent metal ions which are free in solu- tion. In some of the crude extracts the contaminating protein may be able to chelate most of the free metal ions which do not activate the 4-epimerase to any extent, and the more effective cations may be free in solution and thus able to activate the 4-epimerase. However, on purification both the concentration of free metals in solution and the concentration of contaminating proteins changes, and the less activating metal ions will probably be present to compete with the more efficient cations for the active site of the 4—epimerase. DISCUSSION The L-ribulose-S-P 4-epimerase from A. aerggenes with a specific activity of 13 at pH 7.0 and 28°C appeared to be homogeneous as determined by a constant Specific activity before and after crystallization and recrystalli- zation, by a single band on polyacrylamide gel electro— phoresis, and by high Speed ultracentrifugation. The Specific activity of 13 is not a true measure of the poten- tial activity of the enzyme Since this was determined by the continuous assay in the presence of Mg++ and other divalent cations. When Mn++ is the only divalent cation present, a Specific activity of 70 is obtained with the homogeneous enzyme at 28°C and pH 8.0. The L-ribulose-S-P 4-epimerase from A. aerogenagg appears to be similar to the same enzyme purified from E. 3311 (67). Both enzymes exist as a single molecular weight Species on polyacrylamide gel electrOphoresis and on high Speed ultracentrifugation. A molecular weight of 1.14 x 105 for the 4-epimerase from.A. aerogenase is comparable to the molecular weight of 1.04 x 105 which was reported for the enzyme from E, 3211 (67). A Specific activity of 19.0 at pH 7.5 and 37°C was reported for the 4-epimerase from E. ggl$_based on an assay Similar to the continuous assay used here. However, the E. 2213 4-epimerase 142 143 has a pH Optimum between 7 and 10 and, thus, was assayed at its Optimum pH. The effect of divalent metal ion on the activity was not reported. In contrast, A. aerogenes has a pH optimum at 9.0 with 40% of the maximal activity at pH 7.0. However, the pH Optimum may vary depending on the metal ion present. Consequently, it is not possible to compare the activities obtained by the two enzymes. The L-ribulose-S-P 4-epimerases from A. aerogenes and E. ggli were stable to extensive dialysis and chroma- tography on Sephadex G-200, indicating that neither enzyme contained loosely bound cofactors which were not present in the assay mixture. The 4—epimerase from A. aerggenes appears to be activated by Specific divalent metal ions, and the maximal activity obtainable is dependent on the metal ion present, with the order of activation being Mn++)>CO++>>Ni++>>CaI+)> Zn++)>Mg*+. Mn++ appeared to be the most effective of the metal ions since the highest enzyme activity was obtained with this metal, and maximal activity was obtained at the same or lower concentration than the other metal ions tested. Metals from virtually all groups and periods of the periodic table have been found to activate various enzymes; therefore, there may be other metal ions which were not tested which will give higher 4-epimerase activity than Mh++. Metals may participate in enzyme catalysis nonSpeci- 144 fically by creating ionic or electrostatic environments which stabilize the protein, or by complexing with inhib- itors. The metal ion may also be an essential partici- pant in the reaction. Although a metal enzyme may have activity in the absence of metal ion, the presence of the metal ion must change the reaction mechanism. According to Malmstrom (125) a metal ion cannot be said to defini- tively participate in the catalytic reaction unless the influence of the metal ion concentration follows the apprOpriate kinetic parameters, and unless the thermody- namic measurements on interaction of the metal ion with components of the reaction system are consistent with the postulated reaction steps and with the kinetic information. However, in cases where the most obvious artifacts have been excluded and where the rate is greatly increased on the addition of metal ions, it is a reasonable working hypothesis that the metal ion participates in the catalytic reaction. Hence, since (a) L-ribulose-S-P 4—epimerase can be activated over 70 fold by the addition of Mnf'”+ to the metal free enzyme, (b) different divalent metal ions gave varying degrees of activity, and (c) the enzyme was inacti- vated by EDTA and 8-hydroxyquinoline sulfonate, it is reasonable to hypothesize that the metal ion participates in the 4-epimerization reaction. Substantial evidence awaits more thorough kinetic analysis of the metal binding and metal activation prOperties. 145 It is not unreasonable to find that the 4-epimerase is activated by more than one divalent metal ion since most enzymes requiring metals for catalysis are activated by a series of metal ions. The most efficient metal ion is not always the metal ion which gives the highest activ- ity since other metal ions may activate at lower concentra- tions (125). In general metals ions which activate enzymes usually have Similar atomic radii (125). All the metal ions which were tested with the 4-epimerase have Similar radii. There iS no correlation between metal ion activa- tion and coordination number of configuration of complexes either in general or for L-ribulose-S-P 4-epimerase. The theory that EDTA inhibited the enzyme by complex— ing necessary metals is based on the following observations: (a) after removal of free EDTA by passage through a Sepha- dex column, the enzyme was still inactive and could be activated by the addition of metal ions; and (b) EDTA binds Zn++ and Ni++ tighter than Mn"+ yet the enzyme is activated by Mn++ at a lower concentration than that required for Zn++ and Ni++ activation. The fact that the other metal chelators did not inhibit the enzyme is not unreasonable, since most metal chelators are somewhat Specific for certain divalent cations. Thus the chelators may remove some metal ions from solution, but metal ions which are not bound by the chelator are free to bind and activate the 4—epimerase. 146 The possible functions of a divalent catalysis in the metal ion of an enzyme reaction include (126): (a) metal ions may form complexes with donor atoms of either the enzyme or the substrate and thereby enhance their tendancy toward reaction; (b) metal ions may serve merely as a bridge through common coordination to bring the enzyme and the substrate into proximity; (0) while serving function (b) metals may provide as well a chemical activation influence; and (d) while coordinated to either the enzyme or the substrate, metal ions may apprOpriately orient groups undergoing reaction. It should be possible to determine whether the divalent cations are complexing with the substrates or with the 4-epimerase by determining the extent of binding of the metal ion to the 4-epimerase and to L-ribulose-S-P and D-xylulose-S—P. Since Mn++ appears to activate the enzyme, it should be possible to determine the binding constants by nuclear magnetic resonance and electron Spin resonance. An extensive kinetic analysis would also elucidate the role of the divalent cation in 4-epimerization. However, as previously discussed a metal ion would facilitate the enzymatic catalysis of an dealdolization-aldolization reac- tion by acting as an electron Sink, and it would facilitate the prOposed dehydration-hydration mechanism by acting as 41133... 147 both an electron sink, and as a Lewis acid in removing the alpha proton. In addition the metal could facilitate the translocation of the migrating hydroxyl group and could act as a common coordinate to bring the enzyme and the substrate into close proximity. Metal enzymes can contain strongly bound metals and are referred to as metallo enzymes, or they can contain freely dissociable metals and are referred to as metal- enzyme complexes. In order to be classified as a metallo enzyme the metal-enzyme complex must have the following characteristics as outlined by Vallee (127): (a) the ratio of moles of protein to metal must be an integral number; (b) the ratio of metal to coenzyme, when the latter is part of the active complex must be an integral number; (c) the highly purified protein can be isolated with its full metal complement and full activity; and (d) the ratio of moles of metal to protein or coenzyme must be a small number, conforming with the law of multiple prOportionS. Vallee (127) also points out that the characteristics of metal-enzyme complexes include: (a) complete removal of the metal ion may not result in complete abolition of activity; (b) the association con— stant of the metal ion with the reactive group of the protein molecule will be low, and the metal ion will there- fore be readily removed by dialysis; removal will be accompanied by a partial loss of activity and may be 148 restored by the addition of the metal ion; and (c) differ- ent metals may substitute for one another in bringing about activation of the enzyme activity. Since the purified 4—epimerase was not subjected to a metal analysis, it is not known how many of the criteria for metallo enzymes are met. However, it is known that more than one metal can activate the 4-epimerase and activation by Mn++ is partially inhibited by Mgs+. If the inhibition by Hg++ and other metal ions were competitive, it would indicate that Mn++ was readily diSplaced by the other metals, or, in other words, the Mn++ easily dissociates from the enzyme surface and is readily replaced by other metals. If the metal is not tightly bound to the enzyme surface, then 4-epimerase falls into the category of metal-enzyme complexes. This is consistent with the fact that Mg++ and Mn++ usually form weak complexes with proteins (125). A study of the binding prOperties of the different metal ions for L-ribulose-S-P 4—epimerase would also help to determine how the metal ion functions in the catalysis. Of the other 4-epimerases which have been studied only UDP-glucose 4—epimerase (59) and UDP-N-acetylglucos- amine (63) have been reported to be activated by metal ions. The UDP-glucose 4-epimerase activation (59) appeared to be nonSpecific Since the activity was independent of the metal ion added, and activation was also obtained with amines. The metal appeared to prevent product inhibition, 149 and thus the metal may bind to the product and change its prOperties resulting in a shift in the equilibrium of the reaction and a decrease in the amount of product readily accessible to the protein. Glaser (63) reported that UDP-N-acetylglucosamine 4-epimerase was stimulated by Mg++ at a maximal concentra- tion Of 2 x 10'3M and was inhibited by concentrations of Mg++ higher than 1 x 10'2M. The pH Optimum of the enzyme Shifted from 8.5 to 9.5 when Mg++ was removed from the assay. However, the enzyme preparation had only been partially purified, and it was not reported whether the enzyme had activity in the absence of any metal ion or whether other metal ions had any effect on the enzyme activity. Insufficient data were reported to ascertain whether the metal ion was necessary for catalysis or whether the metal ion only had a non-Specific effect on the enZyme. The presence Of Specific divalent metal ions may also Shift the pH Optimum of L-ribulose-S-P 4-epimerase. The Spectrum of the 4—epimerase indicated that the enzyme did not contain any tightly bound cofactors which absorb between 300 mu and 700 mu with a molar extinction coefficient greater than 4 x 102. Thus assuming that cobamide coenzyme, flavins, NADH or pyridoxal phOSphate have approximately the same extinction coefficients when bound to an enzyme as when free in solution, these cofactors could not be tightly bound to the 4-epimerase. Likewise 150 a 280/260 mu absorption ratio of 1.78 is indicative of the absence of a bound adenine moiety. A characteristic reduced NAD+ or flavin fluorescence Spectrum was not pro- duced by the enzyme, and the enzyme was not inactivated by washing with charcoal or incubating with NADase (21). The microbiological assay for NAD+ and the lack of bound 1I+C-»NAD+ on the purified 4-epimerase more defini- tively indicated that L-ribulose-S-phOSphate 4-epimerase does not contain bound NAD+. If NAD+ were bound either covalently or ionically, the entire molecule or the nico- tinamide moiety would have been released by hydrolysis in acid or base. 3. arabinosus would be eXpected to grow on any of the released forms. Hence, the inability of L. arabinosus to use the 4-epimerase as a source of nicotinic acid constitutes strong evidence in support of the theory that NAD+ is not present. In all organisms which have been studied (128), the dg_ngzg synthesis of NAD+ involves the reaction of quinc— linic acid with S-phOSphoribosyl-i-perphOSphate to form nicotinic acid mononucleotide which is then converted to NAD+. Organisms whose dg‘ngzg synthesis of NAD+ is blocked must rely on the conversion of nicotinic acid to nicotinic acid mononucleotide for the synthesis of NAD+. Thus, grow- ing nicotinic acid auxotrOphs in nicotinic acid (carboxyl- 1“0) Should have resulted in the radioactive labeling of all the nicotinic acid nucleotides. The fact that both 151 14 4 C-NAD+ and 1 C nicotinamide could be identified chromato- graphically in the crude extracts indicates that 14C- nicotinic acid was converted to 14C-NAD+ and 1LPG-nicotine amide under the conditions of these eXperiments. The ability to reduce the 1” C content of the 4—epi- merase preparation from an auxotroph grown on 14C—nicotinic acid to less than 0.01 mole of 1”C per mole of 4-epimerase constitutes further substantial evidence that neither NAD+ nor any other nicotinic acid derivative is bound to the 4-epimerase. Further, the 4-epimerase had activity in the absence of added NAD+, and the activity of the enzyme was not increased by NAD+, NADH, NADP+, and nADPH. Thus, it appears that the mechanism of L-ribulose-5-P and D—xylulose-S-P 4—epimerization does not involve a NAD+- catalyzed oxidation-reduction as has been rather substan- tially shown to occur with UDP-glucose 4-epimerase. How- ever, not enough definitive data has been obtained to con— clusively rule out an oxidation-reduction mechanism facili— tated by an enzyme-bound electron acceptor other than NAD+. The electron acceptor would have to be tightly bound to the enzyme surface Since enzyme does not appear to require the addition of any cofactor other than metal ions. It does not seem reasonable that the metal is acting as an electron acceptor. The reduction potential for Mn+++/Mn++ is 1.7 volts removed from NAD+/NADH reduction potential. Since the overall epimerization reaction requires both an 152 oxidation and a reduction step, energy will be released on one Side of the transition step and will be required on the other Side of the transition step, and the net energy change will be zero. Thus, 39 kcal/mole would be released on n+++, assuming the reduction potential of reduction of M the 4—ketOpentose phOSphate to L-ribulose-5-P and D-xylu- lose-S-P was similar to the reduction potential of acetal- dehyde to B-hydroxybutrylaldehyde and assuming the reduc- tion potential of Mn+++/Mn++ is the same when the metal is bound to the protein as when it is free in solution. This same amount of energy would be required to reduce the oxidized intermediate of the pentose phOSphate. Other cofactors which could accept the hydride ion from the pentose-S-P include cobamide coenzyme, cystine, lipoate and the oxidized derivative of tryptOphan as previ- ously discussed. The cobamide coenzyme contains Co++ bond to the 5' carbon of adenine, which is thought to be the hydride accepting site of the coenzyme (105, 106). The Co- carbon bond is readily cleaved by light and acid hydrolysis. Thus, most enzymes containing bound cobamide-coenzyme are sensitive to light and are salmon cOlored. L-Ribulose-S-P 4-epimerase is colorless even in crystalline form and is not sensitive to light, nor does it have an absorption Spectrum characteristic of free cobamide-coenzyme. However, mammaliam methylmalonyl-COA isomerase contains tightly bound cobamide coenzyme and was not inactivated by treatment with 153 charcoal, light, cyanide or extrinsic factor. The enzyme could be resolved by acid treatment, and the apoenzyme was reactivated by dimethylbenzimidazole and benzimidazole coenzyme, but not adenylcobalamine coenzyme (129). Since cobamide coenzyme contains carbon bound Co++, the Co++ is not freely exchangeable with other metal ions in solution. Although the 4-epimerase is activated by CO++, Mn++ is the more efficient metal activator, and the metal ion is freely exchangeable with other metal ions in solution. Cystine and lipoic acid have reduction potentials comparable to that of NAD+ and could, thus, participate in the epimerization reaction. Both disulfide compounds would be active in the oxidized form, and, since disulfides are readily oxidized by air, the oxidized form would pre- dominate on the enzyme surface. Although lipoic acid absorbs at 330 mu, its extinction coefficient was too low to be readily detected. Borohydride should have reduced the disulfide, but, the disulfide may have been reoxidized prior or during the assay. However 4 hours are usually required for air oxidation of cysteine to cystine in pro- tein hydrolysates (130). In addition, enzyme activity was not lost on a one-hour incubation of the enzyme with 1 M mercaptoethanol assayed in the presence of 0.05 M mercapto- ethanol, which are the conditions which are usually suffi- cient to maintain a disulfide in the reduced state. Although 50% of the activity was lost on incubating for an 154 additional hour, the activity was not recovered on passage through the Sephadex column indicating the inactivation was due to some phenomenon other than reduction of a disulfide at the active site. Further arsenite Should have reacted with the reduced disulfide and inactivated the enzyme. Likewise, sulfite Should have reacted with the disulfide forming the stable S-sulfonated derivative and inactivating the enzyme. There are no substantial data to indicate the involvement of an indolenine intermediate of tryptOphan as the electron acceptor in the 4-epimerization. Schellenberg (111) was able to reduce the indolenine salt (o-chloro- phenyl (2-methyl-3H-indolylidine) methane hydrochloride) (I) to the indole (II) by sodium borohydride, dithionite, and 1-benzyl-1,4-dihydronicotinamide. The indolenine salt (I) readily added to mercaptobenzene, benzylmercaptan, and methyl thioglycolate to give the correSponding thioether indole (III). Cl Cl ,/ ~<:f NaBH“ /’ —»CO++)>Ni++:>Ca++>> Zn++)»Mg. EDTA inactivated the 4-epimerase and the activ- ity was not recovered on the removal of the EDTA. Boro- hydride did not inhibit the 4—epimerase either in the presence or absence of substrate. Sulfhydryl reagents such as mercaptoethanol, dithiotheritiol, arsenite, and sulfite, did not inhibit the enzyme at a concentration below 10‘2M. The mechanism of 4-epimerization could be oxidation-reduction, dealdolization-aldolization, or dehydration-hydration; however, the indirect evidence is not consistent with an oxidation-reduction mechanism. 161 10. 11. 12. 13. 14. 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N.,... mm 0.0 H.0H 0.5 0.0 . m N.s 5.5 0.0 0.0 0.0 0.m mm 0.N N.s 0.N :.m 0.0 0.H Aaa.0v a 0H0000HEH 0.0 0.HH 0.5 N.0 mm ) 0.0 0.0 0.0 0.N m.m N.H H.H Hm osfioaauaaoaao N.0H H.mm 0.0a 0.0a H.0H . 0.0 mm efiom 0H000H00-0 s.« 0.NH 0.0 0.0 0.0 0.0 0.0 0.: a mzdaosasumxoo0smum 0.sH 0.0H 0.0m 0.0m 0.Hm 0.00 mm H.NH 0.NH 0.:H 0.3H 0.5 m 3.0 m.o m.w m.m m.m o.m m.o m.a Hm oQHHoHSmesonm1o H.0H 0.0a 0.0H a.ma 0.Ha s.HH m.0 3.0 “0.00 000 Ma H.mH m.mH m.ma s.ma 0.0H 0.0H m.m d.m Ao.mv and M s.0H 0.0a 0.0a 0.0a N.sa 0.00 0.0a a.0 Ha «com o.mm m.mm :.oH mm 0.ma 0.0: «.0 a Ham 2N :0 dz 00 00 ca 00 ma pampmsoo 0502 (in audaapmpm oompmzoo apaafiompm co dog 00 meme Ammav mmNmHoEoo Hmpmz do mummpwmoo apaafinmpm .KHUCmQ94 171