PUREFECATIDN AND CHARACTERIZATIGN Q? A HIGHER MOLECGLAR WElGHT FORM OF YEAST PYRUVATE KINASE Dissertation for the Degree of Ph. D. ,MICHEGAN STATE UNIVERSETY ANN ELEZABEFH AUSF 1975 This is to certify that the thesis entitled PURIFICATION AND CHARACTERIZATION OF A HIGHER MOLECULAR WEIGHT FORM OF YEAST PYRUVATE KINASE presented by ANN E. AUST has been accepted towards fulfillment of the requirements for Ph.D. Jegree in Biochemistry Major professor Date JUly 2’4», 1975 0-7 639 -553 I PURIFICA' The cl a previousl Yeast pyru‘ mechanical. inhibitors in this mar iZe the pyi Procedure. in this ref t0 mechani( 0f the Pot( (DFpl- Th: kinase to l enViIOHmEm aCtivitY 1t fOUnd to r( Contaminan‘ purifiCati( ABSTRACT PURIFICATION AND CHARACTERIZATION OF A HIGHER MOLECULAR WEIGHT FORM OF YEAST PYRUVATE KINASE BY Ann Elizabeth Aust The objectives of this research were: (1) to modify a previously developed purification procedure for bakers' yeast pyruvate kinase so that yeast cells could be mechanically ruptured in the presence of potent protease inhibitors; (2) to determine whether the enzyme purified in this manner was native to yeast; and (3) to character- ize the pyruvate kinase obtained from this purification procedure. The modified purification procedure developed in this research utilized the Manton-Gaulin homogenizer to mechanically rupture yeast at neutral pH in the presence of the potent protease inhibitor diisopropylfluorophosphate (DFP). This homogenizing technique allowed the pyruvate kinase to be more rapidly removed from proteases in an environment containing DFP, which assured a low protease activity level. The Manton-Gaulin homogenization was found to release more pyruvate kinase, as well as other contaminant proteins per pound of yeast. Thus, further purification steps were needed to utilize previously developed precipitat tional cel The enzyme protease a be inhibit that it pr The protec a Sephadex Chara macro- or 1Ytic deg: fied in tl tation tec was Used, ce11.free The immun( adsorbed l 891 elect‘ those of 1 and "natl‘ weight f0 of its m0! PUre PYru enzymeS b The Ann Elizabeth Aust developed procedures. To this end a calcium phosphate precipitation step immediately after lysis and an addi- tional cellulose phosphate column, pH 6.5, were used. The enzyme purified by this means was found to have a protease activity associated with it. The protease could be inhibited by DFP only in 1% SDS solution indicating that it probably existed as a protease-inhibitor complex. The proteolytic activity could be separated from PK on a Sephadex G-100 column. Characterization of the purified enzyme revealed no macro- or micro-heterogeneity, indicative of no proteo- 1ytic degradation. To determine whether the enzyme puri— fied in this manner was native to yeast, an immunoprecipi- tation technique with antibody prepared to the pure enzyme was used. This technique removed "native" PK from the cell-free extract within 5 hours after lysis of the cells. The immunoprecipitate was washed to remove nonspecifically adsorbed proteins and dissolved in SDS for analytical SDS gel electrophoresis. The protein bands visualized were those of purified IgG, used to effect the precipitation, and "native" pyruvate kinase. The subunit molecular weight for the "native" enzyme, calculated on the basis of its mobility in SDS gels, was identical to that of pure pyruvate kinase. This is consistent with the enzymes being identical. The purified enzyme was shown to have a molecular weight of 209,000 by equilibrium sedimentation. Using SDS gel e2 7,500 was tetramer. kinetics : trations l Fructose-i activate ' curve for subsatura‘ FDP activ; velocity shown to I order rat. With incr: With Chan; enzyme Wi‘ that Were COHCEntra. Ann Elizabeth Aust SDS gel electrophoresis, a subunit molecular weight of 57,500 was determined indicating that the enzyme was a tetramer. The enzyme was shown to exhibit cooperative kinetics for PEP, but not for ADP, at saturating concen- trations of all other substrates and metal ions, pH 6.2. Fructose-1,6-diphosphate was shown to heterotropically activate the enzyme, transforming the sigmoid saturation curve for PEP to hyperbolic without affecting Vm. At subsaturating Mg2+ concentration, ADP was found to inhibit FDP activation, producing a time dependent increase in velocity (hysteresis). This hysteretic activation was shown to be a pseudo-first order process. The first order rate of activation appeared to increase linearly with increasing Mg2+ concentration, but remained constant with changing enzyme concentration. Incubation of the enzyme with FDP before assaying produced final velocities that were greater than those without preincubation for PK concentrations greater than 0.5 ug/ml. PURIFICA ir PURIFICATION AND CHARACTERIZATION OF A HIGHER MOLECULAR WEIGHT FORM OF YEAST PYRUVATE KINASE By Ann Elizabeth Aust A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1975 To my family, Brian, Terri, and Steve, and my parents ii I wc Dr. Clare for his a my graduz Yun for r Yeast py] ments to for the l describel the 3min e“JOYed Mark Bro tion, I Biochemi tbrougho of the b FOUUdatj ACKNOWLEDGEMENTS I would like to express my sincere appreciation to Dr. Clarence H. Suelter, my graduate research advisor, for his advice, encouragement, and friendship throughout my graduate work. I would also like to thank Dr. S.-L. Yun for many helpful discussions and for the gift of yeast pyruvate kinase which allowed comparative experi- ments to be done. My thanks also goes to Mr. Guan Ho for the computer program to analyze the hysteretic data described in this dissertation, and to Dr. W. Wood for the amino acid analysis of pyruvate kinase. I have enjoyed many discussions and helpful assistance from Mr. Mark Brody, also in Dr. Suelter's laboratory. In addi- tion, I want to thank the members of the Department of Biochemistry in general for their help and friendship throughout the past five years. The financial assistance of the National Institute of Health, National Science Foundation and Department of Biochemistry is gratefully acknowledged. iii LIST OF '1 LIST OF I ABBREVIAI INTRODUCT LITERATU] Y1 lmrnons rut-3113);? TABLE OF CONTENTS LIST OF TABLES. . LIST OF FIGURES . . ABBREVIATIONS AND DEFINITIONS INTRODUCTION. LITERATURE REVIEW . Yeast Pyruvate Kinase. Activators. ADP . . . . Inhibitors. Reaction Mechanism. . . Cold Lability and Fructose. 1,6- munw> diphosphate Induced Instability . Protease Contamination or Degradation of Yeast Pyruvate Kinase. . . Proteolytic Degradation or Contamination of Enzymes Purified from Yeast Possible in viva Regulation by Yeast Proteases. . . . . . Yeast Proteases. METHODS AND MATERIALS Materials. . . Assay of Pyruvate Kinase Protein Concentration. . . Definition of Unit and Specific Activity . Purification of Pyruvate Kinase. A. Buffers . . B. Preparation of DFP. C. Preparation of Cellulose Ion Exchange Resins . . D. Preparation of Sephadex G- IOO Preparation of Substrates for Assay. Determination of Substrate or Inhibitor Concentrations Kinetic Properties . Polyacrylamide Electrophoresis Assay for Protease Activity in "Pure". Pyruvate Kinase. iv Page vii ix xi HMMHN."’\_. H RESULTS P OI In In Page Isoelectric Focusing . . . . . . . . . . . . 40 Amino Acid Analysis. . . . . . . . . . . . . 41 N- Terminal Analysis. . . . . . . . . . 41 Analytical Ultracentrifugation . . . . . . . 42 Immunization of Animals. . . . . . 0.. . . . 43 Preparation of IgG . . . . . . 43 Ouchterlony Double Diffusion Analysis. . . . 44 Immunoprecipitation of "Native" Pyruvate Kinase from Cell-Free Yeast Extract. . . . 4S ImmunoelectrOphoresis. . . . . . . . . . . . 46 Photography of Results . . . . . . . . . . . 47 RESULTS . . . . . . . . . . . . . . . . . . . . . . 48 Purification of Pyruvate Kinase. . . . . . . 48 A. Lysis of Yeast. . . . 48 B. Ammonium Sulfate Fractionation. . 50 C. Cellulose Phosphate Column, pH 52 6. 5 . . . . . . D. DEAE- CP Column, pH 7.5.. . . . . 52 E. CP Column, pH 6.5 . . . . . . . . 53 F. Sephadex G-100 Column . . . . . . 56 G. Storage of Pyruvate Kinase. . . . 57 H. Criteria of Purity. . . . . 57 Ouchterlony Double Diffusion Analysis of Purified PK and Cell- Free Extract. . . . . 58 Immunoelectrophoresis of Purified PK and Cell- Free Extract. . . . . 58 Immunoprecipitation of PK from Cell- Free Extract. . . . . . 61 A. Enzymatic Characterization of the Immunoprecipitate and Resulting Supernatant . . . 61 B. 1% SDS Polyacrylamide Gel Elec- trophoretic Analysis of Immunoprecipitate . . . . 64 Ouchterlony Double Diffusion Analysis of PK Purified from Yeast Two Different Ways . . . . . . . . . . . . 68 Subunit Molecular Weight . . . . . . . . . . 68 Isoelectric Focusing . . . . . . 82 Molecular Weight of Pyruvate Kinase. . . . . 82 N- Terminal Analysis. . . . . . . . . . . . . 87 Amino Acid Composition . . . . 87 Cold Lability and FDP Inactivation Studies on Pyruvate Kinase . . . . . . . . . 91 Studies on Protease Contamination. . . . . . 95 Kinetic Parameters . . . . . . . . . . . . . 99 Hysteresis . . . . . . . . . . . . . . 100 Cause of Hysteresis. . . . . . . . . . . . 105 Investigation of Inhibitor . . . . . . . . . 105 Investigation of Coupling Assay. . . . . . . 111 V DISCUSS SUBDLARY REFERENI Page Analysis of Hysteretic Curves. . . . . . . 112 Relationship of Velocity and Rate of Activation to Protein Concentration. . . . 114 Relationship of Mg2+ Concentration to Rate of PK Activation. . . . . . . . . . . 119 DISCUSSION. . . . . . . . . . . . . . . . . . . . . 132 SUMMARY . . . . . . . . . . . . . . . . . . . . . . 145 REFERENCES. . . . . . . . . . . . . . . . . . . . . 147 vi Table 10 II 12 Table 10 11 12 LIST OF TABLES Properties of Yeast Pyruvate Kinase Puri- fied by Different Methods. . Properties of Three Purified Proteases from S. cerevisiae . . . . Purification Summary for Pyruvate Kinase from S. cerevisiae . . . . . . . . . . . Enzymatic Characterization of the Immuno- precipitate and Resulting Supernatant. Amino Acid Composition of Manton-Gaulin PK: Determination I . . . . . . Amino Acid Composition of Manton-Gaulin PK: Determination II. . . . . . . . . Amino Acid Composition of Manton-Gaulin PK: Average of Two Determinations Cold Lability and Inactivation of Manton- Gaulin PK. . . . . . . . . . . . . . . . Kinetic Parameters of Manton-Gaulin PK for Substrates and Cations . . . . . Comparison of Manton-Gaulin PK Activity after Preincubation of the Enzyme with Reaction Components. . . . . . . . Comparison of the Ability of ADP Contami- nants or Degradative Products to Inhibit FDP Activation at Subsaturating Levels of Mg2+. . . . . . . . . . Comparison of the Ability of Inorganic Pyrophosphatase to Degrade the ADP or PPi Inhibitor of FDP Activation at Sub- saturating Levels of Mg2+. . . . vii Page 13 27 49 65 88 89 90 93 99 106 107 110 Table 13 14 IS 16 17 Table Page 13 The Ability of Manton-Gaulin PK to Degrade the ADP Inhibitor of FDP Activation at Subsaturating Levels of Mg2+ . . . . . . . . 111 14 First Order Rate Constants and Half Times of Activation for Hysteresis Produced at Different Mg2+ Concentrations. . . . . . . . 129 15 Comparison of the Km Values for Substrates and Cations for Two Preparations of PK . . . 136 16 Comparison of the Properties of PK from Three Different Preparations . . . . . . . . 140 17 Comparison of the Amino Acid Content of PK from S. carZsbergensis and S. cereviaiae. 142 viii Figure Figure 10 LIST OF FIGURES Comparison of SDS electrophoretic mobility of yeast pyruvate kinase before and after Sephadex G-100 chromatography. Ouchterlony double diffusion analysis of Manton-Gaulin PK and yeast cell-free extract. Immunoelectrophoretic analysis of Manton- Gaulin PK and yeast cell-free extract. A comparison of the SDS-polyacrylamide gel electrophoresis profiles of purified immune IgG and the immunoprecipitate formed between yeast cell-free extract proteins and antibody to Manton-Gaulin PK . A comparison of the SDS-polyacrylamide gel electrophoresis profiles of Manton-Gaulin PK and the immunOprecipitate formed between yeast cell-free extract proteins and anti- body to Manton-Gaulin PK . Ouchterlony double diffusion analysis of Manton- Gaulin PK, toluolysis PK, and rabbit muscle PK . . . . . . Determination of the subunit molecular weight of Manton-Gaulin PK using SDS- polyacrylamide electrOphoresis . CompariSon of catalase and Manton-Gaulin PK on SDS-polyacrylamide gels. Comparison of rabbit muscle PK and Manton- Gaulin PK by SDS- polyacrylamide electro- phoresis . . . Comparison of toluolysis PK and Manton- Gaulin PK by SDS- -polyacry1amide electro- phoresis . . . ix Page 55 60 63 67 70 72 75 77 79 81 Figure 11 12 13 14 IS 16 17 18 19 20 21 22 Figure 11 12 13 14 15 16 17 18 19 20 21 22 Isoelectric focusing of Manton-Gaulin PK . Molecular weight determination of Manton- Gaulin PK by high speed sedimentation equilibrium. . . . . . . . . . . . Comparison of active and inhibited pro- teolytic activity on Manton-Gaulin PK using SDS-polyacrylamide electrophoresis Time dependent activation of Manton-Gaulin PK at various Mg2+ concentrations. . . . Time dependent activation of PK in cell- -free extract at subsaturating Mg2+ concentrations Effect of different protein concentrations on hysteresis of Manton-Gaulin PK. . . . Effect of different protein concentrations on the velocity of the reaction for enzyme with and without FDP preincubation Effect of different protein concentrations on the specific activity for enzyme with and without FDP preincubation. . Effect of different protein concentrations on the first order rate constant for enzyme not preincubated with FDP . . Effect of different Mg2+ concentrations on the hysteresis of Manton-Gaulin PK. . + . Effect of d1fferent Mg2 concentrat1ons on first order rate constant . . The relationship between Vf of Manton- Gaulin PK and total Mg2+ concentration. Page 84 86 98 102 104 116 118 121 123 125 127 131 ADP AMP ATP BME CP DEAE DFP DIE DTT EDTA EGTA FDP Igc LDH MantOn- MES NADH NADP PCMB ABBREVIATIONS AND DEFINITIONS ADP ATP BME CP DEAE DFP DTE DTT EDTA EGTA FDP IgG LDH Manton-Gaulin PK MES NADH NADP PCMB adenoSine 5' diphosphate adenosine 5' monophosphate adenosine S' triphosphate Z-mercaptoethanol phosphocellulose or cellulose phosphate diethyl aminoethyl diisopropylfluorophosphate dithioerythritol dithiothreitol ethylenediaminetetraacetic acid ethyleneglycol-bio (B-aminoethyl ether)-N,N'-tetraacetic acid fructose-1,6-diphosphate or fructose- 1,6-bisphosphate immunoglobulin G lactic dehydrogenase pyruvate kinase purified after lysis of the yeast with the Manton-Gaulin homogenizer ZEN-morpholinolethane sulfonic acid reduced nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate parachloromercuribenzoate xi PEP PK PMSF SDS Tetra CI Toluoly Tri CHA Tris PEP PK PMSF SDS Tetra CHA Toluolysis PK Tri CHA Tris phosphoenolpyruvate pyruvate kinase phenylmethylsulfonyl fluoride sodium dodecyl sulfate tetracyclohexylammonium pyruvate kinase purified after lysis of the yeast with toluene tricyclohexylammonium triethylaminoethane xii Lys has prov of the y ferent t Of which intracel Recently that the contaminz to avoid developec Potent PI 17515 Pro Bthmes 0 geneity ( faCt that did not r INTRODUCTION Lysing of yeast for the purpose of purifying enzymes has proved to be a problem because of the rigid nature of the yeast cell wall. Through the years, several dif- ferent techniques have been devised to rupture yeast, most of which are done at elevated temperatures to liberate intracellular proteases as well as other proteins. Recently investigators have become aware of the fact that the liberated proteases were partially degrading or contaminating the enzymes they were purifying. In order to avoid this problem, mechanical methods of lysing were developed which could be used at 4° C in the presence of potent protease inhibitors. In most cases, modifying the lysis procedure in this manner resulted in purified enzymes of larger molecular weight or reduced hetero- geneity (previously reported as isozymes). However, the fact that the enzyme was larger or apparently homogeneous did not remove the question as to whether the purified enzyme was "native" to yeast, for conceivably limited proteolysis could produce an active, homogeneous protein. Investigators, in an attempt to answer this question, have analyzed the purified proteins by a variety of physical methods investil it will identic; 51: method « tion of (Aust e enzyme Because PTEViou in the thought enzYme little in Celt as det identi 1975). tatiOD of PK This n the C1 Donspt the i: Phore 0n SD 2 methods looking for microheterogeneity. This type of investigation will only reveal proteolytic degradation; it will not determine whether the purified enzyme is identical to the native enzyme. Since the discovery that yeast PK purified by the method of Hunsley and Suelter (1969a) resulted in isola- tion of a proteolytically degraded form of the enzyme (Aust et al., 1975), methods for comparing the purified enzyme to enzyme in cell-free extract have been developed. Because the proteolytic damage suffered by PK in the previous preparation did not result in significant changes in the kinetic parameters except in one case, it was thought that a comparison of Km and Ka values for pure enzyme and enzyme in the cell-free extract would be of little value. Analytical disc gel analysis of the enzyme in cell-free extract suggested that the molecular weight, as determined by the Hedrick and Smith (1968) method, was identical to that of the purified enzyme (Yun et al., 1975). This communication describes the immunoprecipi- tation method of comparing the subunit molecular weight of PK from cell-free extract with the purified enzyme. This method involved (I) immunoprecipitation of PK from the cell—free extract, (2) extensive washing to remove nonspecifically adsorbed proteins, (3) solubilization of the immunoprecipitate in 1% SDS solution, (4) electro- phoretic analysis of the solubilized immunoprecipitate on SDS-polyacrylamide gels, and (S) determination and compar from c techni globul 1973), Cullen transp 1973b) 3 comparison of the molecular weight determined for PK from cell-free extract with that of pure enzyme. Similar techniques have previously been used to study immuno- globulins (Baur et al., 1971), O-antigens (Atwell et aZ., 1973), H-Z alloantigens (Schwartz and Nathenson, 1971; Cullen et al., 1972) and rat liver microsomal electron transport proteins (Welton et al., 1973a; Welton et aZ., 1973b). EC 2. LITERATURE REVIEW Pyruvate kinase (ATP pyruvate phosphotransferase, EC 2.7.1.40) catalyzes the following reaction: 2+ + + Mg , K H + PEP + ADP > pyruvate + ATP. t__ This enzyme is ubiquitously distributed as a component of the glycolytic enzyme sequence in all living organisms from simple, single cell organisms to the most complex eucaryotes. Pyruvate kinase has been shown to be an important glycolytic control point in both intact yeast cells and lysates (Hommes, 1964; Pye and Eddy, 1965; Hess and Brand,196Sa). This would be expected since both products of the pyruvate kinase reaction, ATP and pyru- vate, feed into a number of other metabolic pathways, and the substrate phosphoenolpyruvate is a very important controlling compound for carbohydrate catabolism (Kornberg, 1973). The enzyme can be induced in yeast (Barwell and Hess, 1971), and there are reports of probable regulation of the enzyme activity during gluconeogenesis in yeasts (Fernandez et al., 1967). All of these facts strongly suggest that pyruvate kinase plays an important role in yeast metabolism. Sinc Kashio 31 reports I tions of from S. been sho Which wo conditic Preparat 1969a) 1 enZyme Prepara In Yeast e of 0the of Prot YeaSt I \ A' AC1 5 Since the earliest investigation of yeast PK by Washio and Mano (1960), there have been a flurry of reports on the purified yeast enzyme. Original prepara- tions of the enzyme reporting high purity and stability from S. carZsbergensis (Haekel et al., 1968) have since been shown to be contaminated by a protease activity which would degrade the pyruvate kinase under certain conditions (Roschlau and Hess, 1972). The original preparation from S. cerevisiae (Hunsley and Suelter, 1969a) has been shown to produce a degraded form of the enzyme (Fell et al., 1974; Aust et al., 1975) and new preparations were developed to purify the native enzyme. In this review, literature regarding the purified yeast enzyme, literature regarding protease contamination of other enzymes purified from yeast and characteristics of proteases isolated from yeast will be discussed. Yeast Pyruvate Kinase A. Activators 1. Monovalent Cations Pyruvate kinase of S. cerevieiae and of S. carZsbergensie requires the presence of a monovalent cation for activity (Washio and Mano, 1960; Hunsley and Suelter, 1969b; Hess and Haebel, 1967). PK experiences an allosteric activation in the presence of K+; thus, K+ could be referred to as a homotropic activator. Although K+ is tl absence effecti\ Additior intrinsi (Kuczens small cc with K+. TMA. Tl its int: to be tj 9t aZ., EffeCt fluOres to be d Cific K Tl enzymes tains 1 an enz; and 50' {OT th. with t Site ( SPeCif the ac 6 K+ is the most effective activator in the presence or absence of FDP, NH; also activates. Na+ is much less effective and activates only in the presence of FDP. Addition of K+ to PK causes a slight quenching of its intrinsic fluorescence while addition of TMA does not (Kuczenski, 1970). This suggests that PK undergoes a small conformational change as the result of interaction with K+. This change is specific for K+ as opposed to TMA. This would indicate that K+ activation is due to its interaction with the enzyme directly as is thought to be the case in muscle PK (Suelter et al., 1966; Wilson et al., 1967). The binding of K+ to PK has an antagonistic effect on the binding of FDP, as viewed with intrinsic fluorescence changes (Kuczenski, 1970). This is thought to be due both to an ionic strength effect and to a spe- cific K+ effect. The mechanism by which monovalent cations activate enzymes is controversial. One school of thought main- tains that monovalent cations are necessary to establish an enzyme conformation necessary for catalysis (Evans and Sorger, 1966). Another theorizes a specific role for the cations in the mechanism of the reactionzinteraction with the enol-keto tautomers of pyruvate at the active site (Suelter, 1970). The latter theory is much more specific and requires that the cation actually bind at the active site. This has been confirmed in the case of muscle Pl Reuben, ‘ reaction on the 8 PH deper the inte presence of FDP 1 of PEP from 1 at pH 7 Saturat to that to that libriUI lower ‘ state Sugges of Yea and FD 7 muscle PK through the use of NMR spectroscopy (Kayne and Reuben, 1970), but it is not known for the yeast enzyme. 2. Hydrogen Ion Since a proton is taken up in the pyruvate kinase reaction it is conceivable that H+ could have an effect on the enzyme. Wieker and Hess (1971) investigated the pH dependencies of the kinetic parameters characterizing the interaction of S. carlebergensis PK and PEP in the presence and absence of FDP. nH for PEP in the presence of FDP was found to be independent of pH while the nH of PEP in the absence of FDP was dependent upon pH going from 1 below pH 4.0 to a limiting maximal value of 2.95 at pH 7.0. The pH dependence of the ratio of the half- saturating substrate concentration in the absence of FDP to that in the presence of FDP produced a curve similar to that for the n“. These data suggested that the equi- librium between a state of higher affinity and a state of lower affinity of the enzyme for PEP is shifted to a state of higher affinity as the pH approaches 5.35, thus suggesting a role for the H+ as an allosteric activator of yeast PK. The question of whether the H+-activated and FDP-activated states are identical in conformation was not answered. FDP as a The enzy‘ be FDP a a hetero constant the appa Hunsley be class KYIIIaH 31* in bOth earl Mg2+ for PK 31103t6 by FDP, resu1t: Can be anaIYs AinSWQ expres taking ha\re 3. FDP Hess and Brand (1965a) were the first to identify FDP as an allosteric activator of yeast pyruvate kinase. The enzyme from many other sources has also been shown to be FDP activated. In both strains of yeast FDP acts as a heterotropic allosteric activator by lowering the Hill constant for PEP and both required cations and lowering the apparent Km or KA for each (Haekel et al., 1968; Hunsley and Suelter, 1969b). Therefore, PK would best be classified as a K system in the nomenclature of Monod, Wyman and Changeux (1965). 4. Mg2+ Mg2+ has been shown to be a homotropic activator in both S. cerevisiae (Hunsley and Suelter, 1969b) and S. carZsbergeneis (Haekel et al., 1968). In addition Mg2+, or another activating divalent metal, is required for PK activity in both yeast strains examined, and the allosteric kinetics of Mg2+ are transformed to hyperbolic by FDP. The actual form of Mg2+ which binds to the enzyme resulting in catalytic activity is not known. Since Mg2+ can be complexed by PEP, ADP, FDP, and ATP, the kinetic analysis of Mg2+ can be quite complex. MacFarlane and Ainsworth (1972) have developed a complex equilibrium expression for Mg2+ in its free and complexed forms taking into account all substrates and products and have conducted an involved kinetic analysis of PK. Their eva the enzyn released Mgz‘ will be I enzyme c but the by the t Mg2 bergensi Km for ] 1969b) COOpeI-a In the to hth and Su 1968) substr Contrc effeC1 9 2* binds Their evaluation of the results indicated that Mg the enzyme in the free form, as do ADP and PEP, and is released in the complexed form, Mg-ATP. Mg2+ appears to be required for PK stability, as will be discussed later. The binding of Mg2+ to the enzyme can be detected by a quenching of fluorescence, but the quenching is minimal compared to that produced by the binding of FDP (Kuczenski, 1970). Mg2+ appears to affect the affinity of S. earls- bergensis PK for PEP and FDP (Haekel et aZ., 1968). The Km for PEP increases with decreasing Mg. 5. PEP PK from both S. cerevisiae (Hunsley and Suelter, 1969b) and S. carlsbergensis (Haekel et al., 1968) showed cooperative kinetics (homotropic activation) for PEP. In the presence of FDP the sigmoid kinetics are converted to hyperbolic with no change in Vm' B. ADP Kinetic studies of PK from both S. cerevisiae (Hunsley and Suelter, 1969b) and S. carlabergensis (Haekel et al., 1968) indicated that at saturating levels of all other substrates and cations, ADP appeared to be only weakly controlled by FDP and was, therefore, not an allosteric effector for ADP interactions. 10 C. Inhibitors Cu2+ as well as some other heavy metals were shown with early preparations of yeast PK to be potent inhibi- tors (Washio and Mano, 1960). Other inhibitory compounds noted for the brewers' yeast enzyme (S. carZsbergensis), such as citrate, NADP, AMP, 3'-5'-cyclic AMP, and nucleo- tide triphosphates,appeared to exhibit a complex allo- steric inhibition which could be due to their ability to complex Mg2+ (Haekel et al., 1968). ATP inhibition has been observed for both the brewers’ yeast PK (Haekel et al., 1968) and the bakers' yeast PK (S. cerevisiae) (MacFarlane and Ainsworth, 1972). In the case of the brewers' yeast PK, the inhibition appeared to be allosteric, but since no attempt was made to control the Mg2+ concentration throughout the experiment, it would be very difficult to draw firm conclusions. In the case of bakers' yeast PK, the Mg2+ levels were care- fully controlled throughout the experiments and Mg-ATP complex was shown to be a competitive inhibitor with PEP. D. Reaction Mechanism Mildvan et al. (1970) were the first to show incon- sistencies in kinetic and binding data for bakers' yeast enzyme that might indicate that this enzyme has a pre- ferred order of binding PEP. A kinetic study by MacFarlane and Ainsworth (1972) seemed to confirm this, for their results suggested that the mechanism is of the ordered 11 Tri Bi type with the substrates binding in the order PEP, ADP, and Mg2+ An interesting conclusion from this study, apparent in the last statement, was that Mg2+ binds the enzyme in a free form, not complexed to ADP. However, after the phosphoryl transfer in the quaternary complex, pyruvate is released followed by Mg-ATP complex. This posulated mechanism would suggest that Mg2+ binds the enzyme, bridges the phosphate group of PEP and the terminal phosphate group of ADP, assists the phosphorylation of the latter, and is ultimately eliminated, bound between the 8- and y-phosphate groups of ATP. E. Cold Lability and Fructose 1,6-diphosphate Induced’lnstability An instability of yeast pyruvate kinase was first described by Washio and Mano (1960). Glycerol was found to stabilize the enzyme, thus making its purification much easier (Hunsley and Suelter, 1969a). Subsequently Kuczenski and Suelter (1970) reported that pyruvate kinase was susceptible to inactivation at low temperatures, or in other words, that the enzyme was cold labile. Inac- tivation at room temperature and at 0° C was enhanced by the addition of micromolar amounts of the allosteric + + , . 2 or an prevented 1nact1- activator FDP. Addition of Mg vation. A mechanism for the inactivation involving binding of FDP, followed by dissociation of the enzyme into 12 subunits was proposed. Enzyme purified from bakers' yeast by a slight modification of the procedure of Hunsley and Suelter (1969a), produced a cold labile PK, also (Fell et al., 1974). However, PK purified from brewers' yeast (S. carZsbergensis) was reported not to be cold labile (Bischofberger et al., 1971). Protease Contamination or Degradation of Yeast Pyruvate Kinase There have been several methods for purification of yeast PK that have been developed over the past several years. Table 1 shows a summary of some of the important aspects of these preparations and characteristics of the resulting PK. Roschlau and Hess (1972) reported proteolytic con- tamination of purified PK. The activity of the protease was detected noting that free amino acids were released upon storage of the PK at room temperature. Further heterogeneities were seen when PK was subjected to ultra- centrifugal analysis (Bischofberger et al., 1971) and SDS gel electrophoresis. The purification was modified by adding ammonium sulfate fractionation and ion exchange chromatography. The PK obtained was reported free of protease contamination. Fell et al. 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