THE CQNVERSEDN 0F LACTYL 00A T0 ACRYLYL {30A éN PEPTGSTREPTGCGCCUS ELDENN: A. NEW MPHOSPHOLACTYL GOA INTERMEDEATE Thesis for the Degree of Ph. D. MICHEGAN STATE UNIVERSITY DONALD L. SCHNEIDER 1969 u “ - 09m x. IN M \J L 13 If 1"; R E], Michigan State University This is to certify that the thesis entitled THE CONVERSION OF LACTYL COA TO ACRYLYL COA IN PEPTOSTREPTOCOCCUS ELSDENII : A NEW alpha-PHOSPHOLACTYL COA INTERMEDIATE presented by DONALD L. SCHNEIDER has been accepted towards fulfillment of the requirements for Ph.D. dam: in BIOCHEMISTRY flW Major professor Da‘e APRIL 1+. 1969 0-169 =— 1 _, ’ BINDING av ’ HUM; & SONS' 500K BIWQY NE E usmavu «.0: ; - __—--— --—.— ABSTRACT THE CONVERSION OF LACTYL CoA TO ACRYLYL CoA IN Pe tostre tococcus elsdenii: A NEW a-PHOSPHOEKCTYL CoA INTERMEDIATE by Donald L. Schneider Preliminary studies of the direct reductive path- way of prOpionate formation from lactate in g. elsdenii were not consistent with a simple dehydration of lactyl CoA. Thus the existence of an acrylyl CoA intermediate (the product eXpected from dehydration) was reexamined. (1) Lactate was incubated with extracts in tritiated water. The prOpionate which had been produced was found to contain tritium in carbon positions 2 and 3. (2) 3— 3H-Lactate was incubated with extracts in the presence of various amounts of lL’C-acrylate. The prOpionate pro— 3 duced contained lesser amounts of H and greater amounts 14 of C as the amount of 1L"C--acrylate was increased. (3) Lactate was incubated with extracts and acrylyl CoA aminase of lestridium propionicum. Bquanine formation occurred and was dependent on both extracts of g, elsdenii and acrylyl CoA aminase. These eXperiments were inter- preted to mean that acrylyl CoA is an intermediate. At this point the crux of the problem was that ‘ acrylyl CoA is formed from lactyl CoA but not apparently Donald L. Schneider - 2 by a dehydration. An alternate mechanism was considered; the strategy was that since the hydroxyl is a poor leav- ing group. if phoSphorylation occurred to yield c-phos- pholactyl CoA, then the problem of a leaving group would be overcome. The following experiments were employed to test for the possibility of phOSphorylation. (1) 2-180-Lactate was prepared and incubated with extracts. The phOSphate from the mixture was isolated 1'80 content. The results showed that 18 and analyzed for 180 is transferred from 2- O-lactate to phOSphate con- comitant with propionate formation. (2) When lactate was incubated with extracts, the acrylate formed was determined by gas chromatographic analysis. Acetyl phOSphate and catalytic amounts of thiolester (added in the form of acetyl CoA) were required for acrylate formation. Presumably lactate is converted to lactyl CoA by thiolester interchange as catalyzed by CoA transferase; lactyl CoA is phOSphorylated by acetyl phOSphate as catalyzed by a phOSphotransferase; phOSphory- lated lactyl CoA undergoes elimination to form acrylyl CoA as catalyzed by a lyase; and finally acrylate is pro- duced by another thiolester interchange. (3) lac-Lactate and 32P-acetyl pho3phate were incubated with extracts. Analysis of the mixture by paper chromatography of samples withdrawn at various times showed that (a) a 140_ and 32P-labeled compound appeared rapidly, (b) the double-labeled compound had Donald L. Schneider - 3 an RF value equal to that of chemically synthesized phos- pholactate, (c) the level of the double-labeled compound decreased as that of lactate decreased. This pattern was suggestive of an intermediate. (a) The double-labeled compound was partially puri- fied by chromatography on DEAE cellulose and Sephadex G-10. The partially purified material was reincubated with extracts and with added cold lactate. Acrylate and lac- tate were isolated from the reincubation mixture. Determination of Specific radioactivities showed that that of acrylate was greater than that of lactate. This eXperiment was interpreted to mean that the double—labeled compound, which had been formed from lactate, was con- verted to acrylate directly (presumably at the level of thiolester). (5) The partially purified, double-labeled compound was treated with alkaline phoSphatase. Analysis showed that equimolar amounts of lactate and phOSphate had been released. The lactate was tested as substrate for 2? and ‘L58pecific lactate dehydrogenases. Only in the case of Dylactate dehydrogenase was activity observed. Thus the isolated compound is probably a-phospho-D-lactate. (6) Chemically synthesized phOSpholactate was incubated with extracts in order to test whether it would be converted to acrylate. The rate of acrylate formation was found to be 1/10 the rate obtained with acetyl phOSphate and lactate as substrates. The slow Donald L. Schneider - 4 rate may be due to a restricted conversion of phOSpholactate and added acetyl CoA to phoSpholactyl CoA and acetate. In fact without catalytic amounts of acetyl CoA, the for- mation of acrylate from phOSpholactate does not occur. The six eXperiments described above are inter- preted to mean that phOSpholactyl CoA is intermediate between lactyl CoA and acrylyl 00A in the propionate path- way. Thus the conversion of lactyl CoA to acrylyl CoA, which overall is a dehydration, is accomplished by (a) phOSphorylation of the hydroxyl group and (b) B-elimina- tion of the phOSphate. The advantage of this mechanism is the leaving prowess of phOSphate in comparison to the poor leaving prOperties of hydroxide. THE CONVERSION OF LACTYL CoA TO ACRYLYL CoA IN PEPTOSTREPTOCOCCUS ELDENII: A NEW a-PHQSPHOLACTYL CoA INTERMEDIATE By ,i .1, Donald Li Schneider .A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1969 if 2'73 /c:> ACKNOWLEDGMENTS The author wishes to extend his sincere appreciation to Dr. W. A. Wood for his encourage- ment, guidance, and succor throughout the course of this research. The author eXpresses gratitude to his wife, Judith Elnick Schneider, who deepite his lengthy tenure at Michigan State University. maintained faith tantamount to prescience and never remonstrated. His colleagues in the Wood Institute were most kind for tolerating his fre— quent pedantic artisanship. Also the author wishes to thank his parents, Mr. and Mrs. Leonard Schneider, for their approbation. The support of a U.S. National Institutes of Health predoctoral fellowship and an Atomic Energy Commission grant are gratefully acknowledged. ii VITA Donald L. Schneider was born on January 15, 1941, in a purlieu adjacent to Lake Michigan, Muskegon, Michigan. Though obfuscated by instructors of industrial arts and athletics he graduated from Muskegon High School in June, 1959. Whereupon he made a peregrination to Kalamazoo College and from which institution he received the degree of Bachelor of Arts in June, 1963. During the summer of 1962 he had the pleasure of Professor Richard Luecke's tutelage at Michigan State university as a U.S. National Science Foundation under- graduate research participant. As a result he found him- self inSpired to obtain more than a modicum of education. Thus he accepted a graduate research assistantship in the Department of Biochemistry at Michigan State University. Throughout most of the tenure of his Ph.D. work he was supported by a U.S. National Institutes of Health predoc- toral fellowship. The requirements for the Ph.D. degree will be completed in the spring of 1969. Mr. Schneider is a member of the American Chemical Society and Sigma Xi. He is married and without children. In September, 1969, he will commence postdoctoral study with Professor Efraim Hacker at Cornell University. 111 TABLE OF CONTENTS ACMOWLEDGMWTS O O O O O O O O O O O O O O VITA . LIST OF TABLES O O O 0 O O O O O O O O O 0 LIST OF FIGURES O O O O O O O O O O O O O 0 CHAPTER I. II. III. INTRODUCTION 0 O O O O O O O O O 0 LITERATURE REVIEW . . . . . . . . . The Direct Beductive Pathway . . Occurrence and Significance of Direct Reductive Pathway . . Metabolism in P. elsdenii . . . Mechanism of Lactate-Acrylate Interconversion . . . . . . . and E Eliminations . . DehydratIon Properties of Lactate Hydration of Acrylate . . . . Properties of Thiolesters . . _Evidence for CoA Intermediates Evidence for Acrylyl CoA Inter- mediate . . . .Elimination Reactions Involving PhOSphate . . . . PhOSphorylation and Lactyl CoA Dehydrase of P. elsdenii . MATERIALS AND METHODS . . . m . . . Bacteriological . . . . . . . substrates 0 O O O I O O O O O Assays for the Interconversion Lactate and Acrylate . . . Coupled Assay . . . . . Direct Assay . . . . . Manometric Assay . . . Propionate Assay . . . Acrylate Assay . . . . Lactyl CoA Dehydrase Purifi .1 0000000000 09.00. (+0.00. g.» :5 SD iv Page ii iii vii ix CHAPTER TABLE OF CONTENTS (Continued) Other EnZyme Assays . . ' . Organic Acid Purifications Radioactivity Measurements Mass Spectrometry . . . . . Chemicals . . .1. . . . . . Radiochemicals . . . . . . IV. RESULTS 0 C O O I '0 O O O O O O O O O O 0 Confirmation of Lactyl CoA Dehydrase Activity . . . . . . Modified Coupled Assay for Lactyl CoA Dehydrase . . . . . . . . . . . Partial Purification and PrOperties of Lactyl CoA Dehydrase . . . . Evidence Against a Simple Lactyl CoA Dehydrase . . . . . Confirmation of Acrylyl CoA Inter- mediate O O I O O O O O O O 0 O O O The Propionate Assay . . . . . Fractionation of the Enzymes Which Form Propionate From Lactate . . . . Indirect Evidence for Phospholactyl Intermediate . . . .-. . . . . . . Direct Demonstration of a New Inter- mediate. (Presumably d-PhOSpholactyl COA O O O O O O O O O O O Lactyl CoA Kinase Assay . . . . Reversal of DinitrOphenol Inhibition by Acetyl Phosphate o o o o o o o 0 V. DISCUSSION . . . . . . . . . . . . . . . . ABBREVIATIONS USED 0 O O O O O O O O O O O O O O 0 APPENDIX . Calculation of Minimum Specific Activity of the Enzymes of the Acrylate Pathway . . . Calculation of EXpected fiH/lhc Ratio . Stabilization of Extracts by Proteinase Inhibitor I O O O O O O O 0 Lactate Dehydrogenase(s) and Racemase of P. elsdenii . . . . . . . Soluble Electron Transfer System of P. elSdenll o o o o o o o o o Lineari ity of Kcrylate Assays . . . . . Page 40 42 45 46 1+7 49 51 52 '55 59 61 66 75 81 89 132 140 1N6 155 156 156 159 159 162 166 167 TABLE OF CONTENTS (Continued) Page SUMMARY . . . . . . . . . . . . . . . . . . . . . . 171 LITERATURE CITED . . . . . . . . . . . . . . . . . 172 vi Table 10 11 12 13 LIST OF TABLES Lability of Lactyl Thiolester to Storage . Specificity of Coupled Assay for Conver- sion of Acrylyl CoA to Lactyl CoA . . Validation of the Modified Coupled.Assay Using Alcohol Dehydrogenase and Purified. Lactyl GOA . O Q O O O O O 0 Comparison of the Coupled Assays for Lactyl CoA Dehydrase . . . . . . . . Dependence of the Coupled Assay and of the Stability of Lactyl CoA dehyd- rase Upon Dithiothreitol . . . . . . Inhibitors of Lactyl CoA Dehydrase . . . . Tritium Incorporation Into Propionate During Its Formation From Lactate . . Inhibition of the Lactate-to-Propionate Reactions by Added Acrylate . . . . . Chromatographic Demonstration of the Ability of Glutathione to Trap an a,8-Unsaturated.Acyl Ester Inter- mediate (Presumed to be Acrylyl CoA). Requirements for the Conversion of Lactate to Propionate in the Standard ("PrOpionate") Assay . . . . Factors Affecting Stability of "Lactyl CoA Dehydrase" in Extracts of 2. elsdenii O C O O O O O O O O O O 0 Stabilization of Enzymes Converting Lactate to Propionate . . . . . . . . The ActhIty of Calcium PhOSphate Gel Fractions in Converting Lactate to Propionate O O O O O O O O O O O O 0 vii Page 28 54 57 58 6O 64 69 71 73 77 82 83 87 LIST OF TABLES (Continued) Table Page 1# Determination of Order of Function of Calcium PhOSphate Gel'Eluate and Supernatant Fractions . . . . . . . . . 88 15 Inhibition of Propionate Formation and Its Reversal by ATP . . . . . . . . . . 94 16 180 Transfer from Lactate to PhOSphate Concomitant with Propionate Formation O O O 0 O O O O O O O 0 O O O 96 17 Rate of Acrylate Accumulation from Lactate: Effectiveness of Various Acceptors O O O O O O O O O O O O O 0 O 10LF 18 Acrylate Assay Requirements . . . . . . . . 110 19 Conversion of Intermediate to Acrylate . . . 131 20 Alkaline PhOSphatase Treatment of Labeled Intermediate . . . . . . . . . . . . . 135 Appendix Table 1 Lactate Dehydrogenase and Racemase Activ- ities of g. elsdenii Extracts . . . . . 163 2 Divalent Metal Ion Activation of Q-Lactate Dehydrogenase . . . . . . . . . . . . . 16b 3 KM Effect of CoCl on Q-Lactate Dehydro- genase'withI;L-Lactate as Substrate . . 165 A Effect of Ultracentrifugation on Lactate- to-PrOpionate Activity . . . . . . . . 168 5 Non-Linearity of Acrylate Assays with Respect to PrOtein o o o o o o o o o o 169 viii LIST OF FIGURES Figure ' Page 1 Propionate Assay: Specificity for Stereoisomers of Lactate . . . . . . . 79 2 Effect of pH of Storage on Stability of the Enzymes Converting Lactate to Propionate O O O O O O l. O O O O O O 0 81+ 3 Dinitrophenol Inhibition of Propionate Formation: Reversal by ATP . . . . . 91 M Transfer of 32P from y-32PeATP to an Intermediate in the Conversion of Lactate to PrOpionate . . . . . . . . 98 5 Acrylate Accumulation from Lactate: Effectiveness of Various Electron Acceptors I I O O O O O O O O O O O O 102 6 Acrylate Accumulation from Lactate: Effect of Methylene Blue Concentra- t10n................. 105 7 Acrylate Accumulation from Lactate: PhoSphoryl Donor Specificity . . . . . 107 8 Acrylate Formation from Lactate: Requirement for MSClZ o o o o o o o o 111 9 Thin-Layer Chromatography of Reaction Méxture Containing uC-Lactate and P-OrthophOSphate as Substrate . . . 115 10 Separation of 1”C. and 32P-Labeled. Intermediate . . . . . . . . . . . . . 117 11 Transientness of the Compoug d Labeled from Lactate- C and 3EP -Acetyl Phosphate O O O '0 O O O O O O I O O O 120 12 Purification of Enzymatically Synthesized Intermediate on DEAE=Cellulose . . . . 123 13 Purification of Enzymatically Synthesized Intermediate on Sephadex G-10 . . . . 126 ix LIST OF FIGURES (Continued) Figure Page 1h Purification of EnZymatically Synthesized Intermediate on Sephadex G-10 . . . . 128 15 Conversion of the Labeled Intermediate (Which is Contaminated with Labeled Lactate) to Acrylate . . . . . . . . . 132 16 Conversion of Chemically Synthesized d-PhoSpholactate to Acrylate . . . . . 137 17 Lactyl Kinase Assay. The Lactate-Depen- dent Disappearance of Acetyl Phos— phate in the Presence of 10'“ M Dinitrophenol . . . . . . . . . . . . 141 18 Acetyl PhOSphate Reversal of Dinitrophenol Inhibition . . . . . . . . . . . . . . 143 19 The Basic Metabolic System for Lactate Utilization in P, elsdenii . . . . . . 153 Appendix Figure 1 Stabilization of Extracts by a Proteinase Inhibitor . PPISF o o o o o o o o o o o 160 CHAPTER I INTRODUCTION Originally the intent was to study the mechanism of conversion of lactyl CoA to acrylyl CoA as catalyzed by lactyl CoA dehydrase, an enzyme from Peptostreptococcus elsdenii. The mechanism is of particular interest because lactate is difficult to dehydrate by simple chemical means. Further if one considers the reaction in reverse, the hydroxyl group becomes linked to an already electron-rich carbon atom. This is unique in organic and enzymatic reactions inasmuch as a hydroxyl group would be eXpected to add to the beta-carbon. However preliminary experiments raised serious doubts about the existence of lactyl CoA dehydrase: consequently, the purpose of this project is to discover the individual steps and mechanism involved in the conversion of lactate to propionate in g. elsdenii. CHAPTER II LITERATURE REVIEW The most ubiquitous pathway of propionic acid for- mation involves succinate as an intermediate and is called the ”dicarboxylic acid pathway" (Leaver g§_al., 1955; Stadtman and Vagelos, 1957; Wood and Stjernholm, 1961: Swick, 1962). A second pathway involves the direct reduc- tion of lactate to prOpionate via acrylate without any dicarboxylic acid intermediate (Cardon and Barker, 1947; Johns, 1952; Leaver 23 31., 1955; Elsden 22 al., 1956; Ladd, 1957: Ladd and Walker, 1959; Ladd and Walker, 1965; Baldwin gt 3;” 1965). In this pathway lactate-2-140, for example, is converted to prOpionatee2—14C; whereas in the dicarboxylic acid pathway the alpha- and beta: carbon atoms randomize due to the symmetry of succinate. Consequently the dicarboxylic pathway is also called the "randomizing pathway," and the direct reductive the "non- randomizing pathway." THE DIRECT REDUCTIVE PATHWAY glpstridium prOpionicum, an anaerobe, metabolizes lactate, pyruvate, and acrylate to prOpionate, and these fermentations constitute the first evidence of the occur- rence of the direct reductive pathway (Cardon and Barker, 2 3 1947). 2;. prOpionicum is not able to decarboxylate suc- cinate and is not able to ferment malate or fumarate (Johns, 1952). Furthermore, lactate-B-luc is fermented to propionate-3-1uc by whole cells (Leaver 22 31., 1955). These observations clearly eliminate succinate as an intermediate and are consistent with the direct reduction of lactate. The propionate = acrylate reaction which is catalyzed by g1. prOpionicum extracts occurs at the level of CoA thiolesters (Stadtman and Vagelos, 1957). Such evidence suggests that acyl thiolesters might be the intermediates in the direct reductive pathway: fi if f: C-SCoA C-SCoA C-SCoA | -H20 | 2 H I HOCIIH : is > 1:32 CH3 CH2 H3 9 PEPTOS‘I'RH’TOCOCCUS ELSDEN II The most thoroughly studied organism possessing the direct reductive pathway is Peptostreptococcus elsdenii. It is a Gram-negative, strict anaerobe which was isolated from the rumen of sheep (Elsden gtflgl., 1956) and cow (Gutierrez gt_g;., 1956). It ferments acrylate without carbon dioxide fixation, thus ruling out the dicarboxylic acid pathway; furthermore acetone powders are able to utilize acrylate to form propionate and acetate (Lewis and ElSden . 1955) o h g. elsdenii also ferments lactate, glucose, fruc- tose, and maltose (Gutierrez gt 31., 1956); but is not able to ferment succinate, malate, or fumarate (Elsden 33 31., 1956). Other general prOperties are (1) lack of Spores, (2) non-motility, (3) evolution of carbon dioxide and hydrogen, (h) formation of fatty acids up to g- hexanoate (Elsden e§,§l., 1956), and (5) formation of acetate by the phOSphoroclastic reaction (Peel, 1960). The name, Peptostreptococcus elsdenii, was given on basis of the following characteristics (1) chain formation, (2) rapid fermentation of carbohydrates, (3) coccal mor- phology, and (h) ability to attack organic acids (Gutierrez gt_gl., 1956). Additional support for the direct reductive path- way in P, elsdenii is that lactate-Z-luc is fermented to prOpionate with the label exclusively in the methylene carbon (Ladd, 1957). These results were confirmed with extracts (Ladd and Walker, 1959). Lactate and acrylate are fermented by cell-free extracts at identical rates and to the same products in identical proportions (Ladd and Walker, 1959). Lactate activation by thiolester for- mation seems to be necessary because, after five hours of dialysis, the addition of ATP or a source of active phos- phate is required in order to restore the ability to form propionate. As in 2;. prOpionicum, acrylate reduction requires prior thiolester formation (Ladd and Walker, 1965). 5 Further suggestive evidence for thiolester activation is that, when lactate-14C is incubated with extracts of P. elsdenii, the hydroxamic acids of lactate, propionate, acetate, and perhaps acrylate can be identified (Ladd and Walker, 1959; Baldwin g§,§l., 1962; Baldwin §£.§l., 1965). All enzymes of the pathway have been demonstrated in P, elsdenii (Baldwin, 1962; Baldwin e§_gl., 1965). CoA transferase activates lactate by catalyzing a general thiolester exchange with, for example, propionyl CoA: (1) lactate + propionyl CoA = lactyl CoA + propionate. (2) lactyl CoA == propionyl CoA. NET REACTION: lactate = propionate. The acyl CoA dehydrogenase and lactyl CoA dehydrase were also demonstrated and partially purified. OCCURRENCE AND SIGNIFICANCE OF DIRECT REDUCTIVEvPATHWAY As already discussed, the pathway was first observed in.gL,propionicum. It occurs in P. elsdenii and, in this case, contributes to bloat, a disorder in cattle character- ized by distention of the rumen and colon. In a cow afflicted with bloat g, elsdenii becomes very prominent as the microbial population changes. Its association with bloat probably is based on evolution of large quantities of gas, namely carbon dioxide and hydrogen (Gutierrez 23 51., 1956). 2, elsdenii exemplifies the importance of the pathway in another way. As the readily-available carbo- 6 hydrate in a cow's diet increases, so does the population of P, elsdenii in the rumen. Labeling eXperiments show that rumen microorganisms form 70-100% of their prOpionate by the direct reductive pathway (Baldwin, 1962; Baldwin gt 21.. 1963). The pathway has been well-documented to occur in legume nodule bacteroids (Jackson and Evans, 1966). Bac- teroids produce propionate which is then utilized by the plant, e.g., for heme biosynthesis. Dialyzed extracts of bacteroids require ATP, Mg2+ , and NADH as cofactors to convert lactate to prOpionate. The fourth known occurrence of the pathway is Bacteroides ruminocola (Wallnbfer and Baldwin, 1967). Apparently this organism, unlike P. elsdenii, becomes pre- dominant when the cow's diet does not readily furnish car- bohydrates, e.g.. hay. A Most known sources of the pathway consist of strictly anaerobic bacteria which are symbiotic. Whether the pathway occurs in plants or animals has not been thoroughly tested, though pigeon heart muscle has been found to possess acrylyl CoA hydrase activity. In this case however, the reaction is irreversible and produces lactyl CoA (Vagelos‘gt‘gl.. 1959). The pathway may func- tion in reverse to form lactate in Moraxella lwoffi (Hodgson and McGarry, 1968), Escherichia ggli (Wegener gtugl., 1967), and in Pseudomonas aeruginosa (Sokatch, 1966). METABOLISM IN P. ELSDENII The fermentation products of P. elsdenii grown on lactate are acetate, propionate, C02, H2, and lesser amounts of butyrate, valerate, and hexanoate. Inasmuch as anaerobes, by definition, cannot reduce oxygen to water, electrons which are obtained by oxidation of metabolites in the course of providing high energy compounds are accepted by molecules also formed in the process, e.g.. in this case acrylate, crotonate, etc. In otherwords, prOpionate is formed as a result of acrylyl CoA accepting electrons which are obtained by oxidation of lactate to acetate: 3 LACTATE > 2 ACRYLATE ADP+P1 4e“ 1 ACETATE + C021 + ATP 2 PROPIONATE Lactate is oxidized first to pyruvate by NAD- independent dehydrogenase(s) and then to acetate and car- bon dioxide by means of the phOSphoroclastic reaction (Baldwin, 1962). Since the latter reaction requires phos- phate, elimination of phosphate would block oxidation of pyruvate. Under this condition lactate is oxidized to pyruvate with concomitant reduction of acrylyl CoA to propionyl CoA, i.e., all electrons from oxidation of lac- tate to pyruvate are transferred exclusively to acrylyl CoA and do not contribute to hydrogen formation (Ladd and 8 Walker, 1959). Though.P, elsdenii in the laboratory pre- dominantly encounters L—lactate, its lactate dehydrogenase is specific for the Deisomer (Baldwin, 1962). An eXplana- tion may be the prior conversion of‘L- to Dylactate via a racemase. Such racemases have been found in other organ- isms: Clostridium acetobutylicum secretes a Bé-Fe2+-depen- dent racemase into the medium which is thought to catalyze dehydration and rehydration of lactate (Katagiri 22 21., 1958): 9;. butylicum produces a racemase with an Splactyl intermediate and an internal hydride shift involved in the mechanism (Dennis and Kaplan, 1959). The Delectate dehyd- rogenase of g. elsdenii has been partially purified (Baldwin, 1962). The phoSphoroclastic reaction oxidizes pyruvate to acetyl phOSphate and carbon dioxide and is of the clos- tridial type, i.e., pyruvate decarboxylase forms CO2 and hydroxyethylthiamine pyrophoSphate (TPP); the hydroxy- ethlePP is oxidized by ferredoxin to acetyl TPP: acetyl TPP reacts with COASH to regenerate TPP and to produce acetyl CoA: phOSphotransacetylase has been partially puri- fied (Baldwin, 1962) and catalyzes the formation of acetyl phoSphate from inorganic phOSphate and acetyl CoA, whence the term "phoSphoroclastic" reaction (Ladd, 1959; Peel, 1960; Joyner and Baldwin, 1966). Supposedly acetyl phoSphate is the organism's source of energy. The substrate-level phOSphorylation 9 catalyzed by acetokinase would produce one mole of ATP for each mole of lactate oxidized. The fate of reduced ferredoxin is two-fold. In extracts, a powerful hydrogenase accepts its electron to form % H2 in a reaction which is predominant below pH 7.6 (Ladd and Walker, 1965). Since the rumen is slightly acidic, the electrons from ferredoxin would be eXpected to be used to evolve hydrogen in nature; instead, in whole cells these electrons are used in formation of higher fatty acids. At slightly alkaline pH the ferre- doxin electrons can be transferred to acyl CoA dehydro- genase by way of NAD and an electron carrying protein (Baldwin and Milligan, 1964). Thus in extracts the fer- mentation normally is: ADP + 2 lactate = acetate + 002 + ATP + H2 + propionate, and at pH's above 7.6 is: ' ADP + 3 lactate = acetate + CO2 + ATP + 2 propionate. Notice that the small amounts of higher fatty acids have been omitted from the above formulation. The acyl CoA dehydrogenase has been partially puri- fied and may contain a cytochrome cofactor. Coupling of lactate oxidation with acrylyl CoA reduction has been cal— culated to afford -AF = 18 Kcal/mole which is more than enough for formation of a high energy phOSphate bond (Barker, 1956). Furthermore P, elsdenii grows very well for an anaerobe with growth yields as high as 10 g of wet cells/l. Electron tranSport phOSphorylation may occur in 10 2, elsdenii: however, employing partially purified acyl CoA.dehydrogenase and electron carrying protein, ATP for- mation concomitant with that of propionyl CoA was not demonstrable (Baldwin and Milligan, 1964). The formation of fatty acids higher than propion- ate has not been studied, undoubtedly because extracts are not very active in this regard (Ladd and Walker, 1959). 1LLC-labeling eXperiments have shown, however, that butyr- ate is synthesized from two acetates, hexanoate from three, and valerate from one acetate and one prOpionate with the prOpionate moiety occupying carbon positions 3, 4, and 5 (Ladd, 1957). CoA transferase enables g, elsdenii to conserve the energy of active thiolester intermediates and is Specific for the CoA moiety, rather non-Specific for the acyl group, and has been partially purified (Baldwin, 1962). In this manner lactate may be activated without expenditure of ATP: propionyl CoA + lactate = lactyl CoA + prOpionate. MECHANISM OF LACTATE - ACRYLATE INTERCONVERSION Lactate dehydration requires the elimination of the elements of water. Elimination reactions are known by the organic chemist to occur via two mechanisms (1) unimolecularly and (2) bimolecularly with an assisting molecule of base. The unimolecular elimination is called 11 E1 and involves as a first step the removal of a hydroxyl group to form a carbonium ion: O O _ O I ll Ll C-SCoA C-SCoA -SCoA l e - l rds I HOCH——) HO + @CH ———> + lclm CH3 H3 CH2 . In this instance the reaction is absolutely improbable because the strong electron withdrawal effects of the acid carbonyl and the CoA thiolester would greatly reduce the stability of this carbonium ion. Indeed the CoA is sufficiently electron withdrawing to stabilize an 2122.“ carbanion to the extent of lowering the alpha-hydrogen's pKa 4 units below that of the parent acid (Lynen, 1953). This will be discussed more thoroughly below. ' The bimolecular eliminations are called E2 and are dependent on a good leaving group, X, and acidic hydrogens: O 0 II I' C-SCoA C-SCoA 6 HO-CH a I-IB-enz + H0. + ICIH CH-H*—‘~:B-enz CH 2 2 . Clearly the E2 mechanism is the more reasonable of the two; nevertheless two difficulties exist. In the first place a beta-hydrogen is not nearly as acidic as an alph - one at least in the case of lactyl CoA. Second, the 12 hydroxyl is a poor leaving group (Gould, 1959). In fact lactic acid is difficult to dehydrate chemically: when ‘alphgrhydroxycarboxylic acids are subjected to dehydrat- ing conditions (strong acid, heat) they form cyclic dimers: R R é I H CH HO’/ \‘C=O O’/ C=O HO OH H69 g + 2 H20 0 A OH “ O C \\\ \\\ // Hf HF R R (Morrison and Boyd, 1959). Concentrated lactic acid catalyzes the reaction itself and thus lactic acid always contains significant amounts of the dimer, called lactide. alpha-Hydroxyacids are dehydrated to QIEQEJQEEE- unsaturated acids industrially by use of heat and cata— lysts among which sulfate and phOSphate are common (Ustavshchikov 23 21., 1965; Holmen, 1958). Lactate and its oxygen esters have been dehydrated in this fashion. Perhaps these catalysts work by first forming an ester with the hydroxyl group: 0 COOH O COOH T T (3 O-P-O + HOCH ___. eO-P-O-CH + OH 9 (I) O CH I C> O CH 3 C) 3 13 and by eliminating E2 fashion: O O COOH O-ITD-O COOH T ,I e | e l O-P- -CH 0 CH 0 €>$ a} C) II T e .\ (T) CH2 HO—IID-Oe . CH2.- rQD-P-Oe 09 I 0C) The principal advantage of this scheme is that phOSphate is a much better leaving group than the hydroxyl group as will be discussed below. Consider the dehydration of lactate in reverse. Since acrylate is involved, its chemical prOperties as well as those of acrylyl CoA are important. Acrylic acid undergoes addition reactions in anti-Markownikoff fashion, e.g., hydrogen bromide yields beta-bromoprOpionic acid. The first step in the reaction is transfer of a proton to form a resonance hydrid of the following structures (Roberts and Caserio, 1965): CH -CH-COOH + H® 2- 3 @OH OH OH _ ,/ _ _ /’ _ _ // CH2..CH- \OH H CH2_CH C§OH <—-> @CHZ CH_C\OH G C In keeping with this reaction, in which the more electron- withdrawing carboxyl group is protonated rather than the double bond, bromine reacts heterolytically very slowly with acrylic acid on account of the absence of a proton 1n (Fieser and Fieser, 1956). Acrylic is a stronger acid than prOpionic: Acid pKa Ref. lactic 3.87 Fieser and Fieser, 1956 acrylic 4.26 " acetic 4.76 " propionic 4.88 " Since the properties of free acrylic acid differ from those of saturated acids, acrylyl CoA is eXpected to be unusual too. A discussion of thiolesters follows. In a thiolester pn-dn orbital overlap might be eXpected (resonance form 1) making the C-8 bond stronger; however this form is not significant (Bruice and Benkovic, 1966a). O OO 069 II I G | -C-8- 6—) -C=S- H -C-S- GD 1 2 The low electronegativity of sulfur does give resonance form 2 greater importance than in oxygen esters (oxygen 3.5; sulfur 2.5 (Pauling, 1960)). Nevertheless both resonance forms would (1) increase the acidity of the alpha-hydrogens of a thiolester, and (2) assist nucleo- philic diSplacement at the carbonyl carbon. 15 Thiolesters are much more reactive than oxygen ones; Lynen classifies them as acid anhydrides. The acidifying effect of sulfur can be measured in acetoacetic esters by observing the dissociation of a methylene hydrogen (see below). This activation of the alph_-hydrogens accounts for the normal reactions of thiolesters, e.g., acetyl CoA and oxaloacetic acid react to form citric acid as though the alpha-carbon atom of acetyl CoA were a carbanion. Compound pKa Reference acetoacetic acid 12.70 Lynen, i953 ethyl acetoacetate 10.70 “ Seacetoacetyl-flgacetylthioethanolamine 8.50 " Thiolesters which are alph§,bgtg-unsaturated absorb at 22k mu, whereas the saturated ones absorb at 204 mu. The shift to longer wavelengths is probably due to double bond-C0 group resonance, which may eXplain why unsaturated thiolesters behave like alEQEJQEEE-unsaturated ketones towards reducing agents, e.g., leuco dyes. Furthermore a new band appears at 263 mu which might be due to n-inter- action between the double bond and the -S-C0 group. Since the 263-mu-absorbance peak is unique to unsaturated thiol- esters it offers a means of measuring any reaction involv- ing acrylyl CoA (Lynen, 1953). Acrylyl CoA is more reactive in addition reactions 16 than the free acid, e.g., SH groups will add across the double bond of the ester whereas the acid is unreactive (Dixon and Webb, 1964). In perSpective then the chemistry of acrylyl CoA clearly suggests that in an addition reaction the anionic moiety should go beta because the alpha-carbon is more electron-rich. Thus acrylyl CoA to lactyl CoA is, mechan- istically, an uneXpected and unusual reaction which merits study and stands in contrast to the normal reactions of acrylyl CoA, e.g., adding an SH group to form 3-thiol- prOpionyl CoA or an amino group to form.bgt§-alanine (cata- lyzed by acrylyl CoA aminase). Consider again the evidence for CoA intermediates: (1) acrylate reduction in 9;, prOpionicum requires prior thiolester formation, (2) lactate or acrylate fermentation to propionate by E, elsdenii requires catalytic amounts of ATP or thiolester, (3) the radioactive hydroxamates of lactate, acetate, propionate, and perhaps acrylate can be isolated when lactate-1n C is fermented by g, elsdenii, and (4) all the enzymes of the pathway have been demon- strated in g. elsdenii, and all involve acyl CoA substrates (Baldwin g£.g;., 1965). In fact the coupled assay, which was developed for the key enzyme, lactyl CoA dehydrase, shows an absolute Specificity for acrylyl CoA (Baldwin 22 El., 1965: Baldwin, 1967). The evidence for thiolester intermediates seems rather clear-cut, but that for an acrylyl one is less so. 17 Previously, enzymes which replaced a hydroxyl group with a hydrogen atom were believed to (1) eliminate water to form a double bond and (2) to reduce the double bond: OH I ~H20 2H. -C-C- ; -C=C- I | l H H H However this generalization is no longer universal. In the case of deoxycytidine diphOSphate formation, the con- cept was thought to apply (Beichard, 1962); and recently the mechanism has been shown to involve hydride ion-like displacement of the 2'-hydroxyl group of cytidine diphos- phate (Larsson, 1965; Durham.g§_§;,, 1965). With this in mind the evidence for acrylyl CoA as an intermediate must be scrutinized anew. As early as 1942 it was written "0f several mechan- isms pr0posed, that involving removal of water from lactic acid to form acrylic acid which is then reduced to pro- pionic acid has seemed the most probable" (Werkman and Wood, 1942). DeSpite these eXpectations the evidence for an acrylyl intermediate remains suggestive, though its existence seems probable. Each datum supporting an acrylyl intermediate is discussed below. (1) Acetone powders of g, elsdenii reduce acrylyl CoA to propionyl CoA (Lewis and Elsden. 1955). However this activity could be due to the acyl CoA dehydrogenase of fatty acid synthesis (enoyl reductase). Indeed this (2) (3) 18 was shown to be the case in prOpionibacteria in which l”c (Swick, acrylate-14C is converted to succinate- 1962). Extracts of g, elsdenii ferment acrylate at the same rate and to the same products as lactate (Ladd and Walker, 1959). This is good evidence, but one could again argue that the fatty acid synthesizing acyl CoA dehydrogenase reduces acrylyl CoA to propionyl CoA which then shuttles back through the direct reductive pathway to lactyl CoA. Such an explanation would require that those enzymes be more active than the enzymes involved in lactate to acetate; the analysis of the end products of the fermentation suggest that this is probably the case (Gutierrez 32 31., 1956). Extracts of E, elsdenii catalyze the hydration of lac-acrylyl CoA to 1” C-lactyl CoA (Baldwin 23 gl.. 1962). However, since the identification involved paper chromatography of the hydroxamic acids and since acrylyl hydroxamate polymerizes, it can give Spots all over the chromatogram from RF 0.65 (unpoly- merized, same as propionate) to RF 0.20 (Baldwin gt 2;., 1965). Other investigators suggest that acrylyl hydroxamate polymerizes so readily as to give R 0.00 F (Ladd and Walker, 1959). Since acrylyl hydroxamate polymerizes the identity of any Spot is uncertain. Furthermore, even if luC-lactyl CoA is actually pro- duced, the fatty-acid-synthesis acyl CoA dehydrogenase l9 possibility, and subsequent reaction of propionate to lactate may apply (of. above (1)). (4) In a coupled assay system involving the reactions 14 written below, the formation of C-pyruvate is depen- dent on enzyme, NAD, LDH, and 14C-acrylyl CoA (Baldwin 23 920 g 1965): 15-fold pur. enzyme 1) acrylyl CoA + H20 A;lactyl CoA CoA transferase 2) lactyl CoA + acrylate : lactate + acrylyl CoA LDH 3) lactate + NAD ; pyruvate + NADH diaphorase 4) NADH + INT >NAD + INTL’85 m“ reduced' NET: HZO + acrylate + INT.___;pyruvate + INTreduced' With this assay, the enzyme could be purified fifteen- fold before activity vanished. However the purified enzyme could not be assayed successfully in a direct manner utilizing the absorption of unsaturated thiol- esters (Lynen, 1953). This could be due to product inhibition, i.e., acrylyl CoA might bind to the enzyme and prevent recycling of the enzyme until the product is consumed in a subsequent reaction. 0n the other hand, the direct assay has been used for other enzymes which catalyze the acrylyl CoA = lactyl CoA reaction (Vagelos gt‘gl.. 1959). (5) The fifteen-fold purified enzyme was shown to be rever- sible by using acyl CoA dehydrogenase and reduced 20 i4 safranine as a coupling system and C-lactyl CoA as substrate (Baldwin, 1962; Baldwin 23 31., 1965): 4 enzyme 14 1) 1 C—lactyl CoA : C-acrylyl CoA + H20 4 acyl CoA dehyd- 2) 1 C-acrylyl CoA + reduced safranine ; rogenase 14 safranine + C-propionyl CoA. NET: luC-lactyl CoA + reduced safranine._______9 14 320 + The difficulties are that (1) a fifteen-minute preincu- C-propionyl CoA + safranine. bation of the components of reaction 1) is necessary and is uneXplainable, and (2) the direct assay of this same reaction does not work. Two groups have reported that acrylate interferes with the conversion of lactate to prOpionate (Jackson and Evans, 1966; Whanger and Matrone, 1967). Though the obser- vation is consistent with an acrylyl intermediate, it is, unfortunately, also consistent with acrylate as an inhibitor. Nonetheless the evidence taken all together, makes the case for acrylyl CoA as an intermediate suggestive. At the same time alternate reactions leading to prOpionate must be kept in mind. One mentioned previously is hydride diaplacement as seen in deoxycytidine diphOSphate biosyn- thesis. Another might involve activation of the hydroxyl of lactyl CoA prior to dehydration, e.g., phOSphorylation. PhOSphate is a much better leaving group than hydroxyl and in the presence of a beta-electron-withdraw- 21 ing group leads to base catalyzed beta-elimination. Some examples are phOSphoserine, 2-cyanoethyl phOSphate, 2- sulfoxyethyl phOSphate, glyceraldahyde-B-phOSphate, adenosine-5'-phOSphate, and fructose-1,6;diphOSphate (Bruice and Benkovic, 1966b). The elimination of phos- phate may be much easier than hydrolysis, e.g., phOSphO- lactic acid under Optimum conditions for each would be eXpected to form acrylic acid about 500,000 times faster in alkaline solution (pH 14) than it forms lactic acid in acidic solution (pH 4.5) (Cherbuliez gt_gl., 1962): 0 c009 O 0009 T ol 9 Hfi |H 9: O-fi-O + H 4- HOH . C“DOG"? a? H-""@OH 09 £132 Enzymatic precedent for the aforementioned phOSphate- facilitated elimination is mevalonic acid perphOSphate decarboxylase (ATP: 5-perphOSphomevalonate carboxylyse 4.1.1.33). ATP and Mg2+ are cofactors; the enzyme is involved in steroid biosynthesis (Bloch gt_§l., 1959; Henning gtflgl., 1959; Waley, i962): CH2-0® O® CH20®0® (IJHz-0®O® CH CH CH l 2 ATP 2 -C02 Cl 2 HO-C-CHB >-®-0 c 033 2-; C-CH3 I -HPOu " (:32 {:32 CH2 0009 c 0% \09 MEVALONIC ACID ISOPENTENYL PYROPHOSPHATE PYROPHOSPHATE. 22 Another example is threonine synthetase in E. coli (Flavin and Kono, 1960): 000' C00 HzN-CH 18 HZN-CH | H2 O | 18 l $32 CH3 O QrPHOSPHOHOMOSERINE THREONINE If the reaction is carried out in 32180, one atom of sol- vant oxygen is incorporated into threonine and none into phOSphate. Cleavage occurs at the C-0 bond of the phos- phate ester, and the phOSphate is formed by nonhydrolytic elimination. The biosynthesis of uridine diphOSphofiNgacetyl- muramic acid demonstrates the leaving prowess of phOSphate though a diSplacement rather than an elimination is involved (Gunetileke and Anwar, i968): COOH CH OH CH OH I 2 o 2 O P CC + UDP UDP + P . “ HO H HO 1 CH2 HNAc NAc O ‘\fOOH PEP UDP-Glc-NAc é 23 Suggestion for phOSpholactyl CoA and/or electron transfer phOSphorylation in g, elsdenii is that the inter- conversion of acrylyl CoA and lactyl CoA is prevented by uncouplers of oxidative phOSphorylation, e.g. dinitrOphenol (Ladd and Walker, 1959). Yet the system contains no mito- chondria and is particulate free. These workers hint at the existence of phOSpholactyl CoA as they write (Ladd and Walker, 1965), "... the interconversion of lactyl CoA and acrylyl CoA is not the simple reaction shown, but requires a source of energy geared possibly to the transfer of electrons from lactate to acrylyl CoA." Certainly this is in contrast to more commonplace dehydrases, such as 6— phOSphogluconic acid dehydrase which requires a divalent metal and free thiols (Meloche and Wood, 1964a). If lactyl CoA is phosphorylated there must be a source of the active phOSphate, and since 2, elsdenii is an anaerobe this may be a serious problem. The phOSphoroclastic reac- tion is used for energy production and cannot be considered as the source in this case, eSpecially since the fermenta- tion analysis shows that the direct reductive pathway is more active than the elastic reaction (Gutierrez 23.31,, 1956). Another possibility exists, and that is electron tranSport phOSphorylation. Some anaerobes grow beyond the limits of their known substrate phOSphorylation, and, at least in the case of 2;. aminobutyricum, electron tranSport phOSphorylation has been demonstrated to occur during the reduction of crotonyl thiolester (Hardman and Stadtman, 24 1963). This phOSphorylation is feasible on a thermodynamic basis because there is a negative free energy change of 18 Kcal under physiological conditions (Barker, 1956). Whether this occurs in g, elsdenii is difficult to test directly because of an extremely active myokinase. CHAPTER III Wig: AND METHODS BACTERIOLOGICAL A culture of g, elsdenii (ATCC no. 17752; strain B 159) was the kind gift of Professor M. Bryant. The organ- ism was maintained in stock culture as described by Elsden and Lewis, except 0.001% resazurin was added as a redox indicator (a red color indicates lack of anaerobiosis) (Elsden and Lewis, 1953). The stock culture has variable viability after prolonged storage and in order to elimin- ate the danger of losing the culture it was transferred every two weeks. From time to time Gram stains were made to determine cultural purity (Conn, 1957); contaminants were removed by plating either on thioglycollate media with 2% sodium lactate solution (60%) added in ordinary plates in a desiccator under N2 or on stock culture media with 1% agar added in rolled tubes according to published procedures (Hungate, 1950). When plating, the medium was cooled to 45°C and inoculated; the medium was immediately poured into plates under N2. g, elsdenii was grown in deep culture on corn steep liquor and lactic acid as described previously (Ladd and Walker, 1959) except that distilled water was employed and trace metals were added 25 26 as described for a defined medium (Allison gt_§l., 1966; Bryant and Robinson, 1961); however two attempts to grow 3. elsdenii on this defined medium were not successful. Extracts were prepared by suSpending the frozen cells under N2 in an amount of buffer in milliliters equal to their weight in grams. The buffer was 0.1 fl_phOSphate (K+) (pH 6.5) and contained 1 mg DTT as reducing agent. The cells were disrupted by either (1) two passages through a French pressure cell or (2) 10 Kc sonication for 20 min. The temperature during disruption was 0-5°C and DNase was added to extracts prepared by the French press. The extracts were stored at -14°C under N2. Addition of the proteinase inhibitor phenylmethylsulfonyl- fluoride as used by others (Steinman and Jakoby, 1967) was in some instances beneficial for stability. SUBSTRATES Acetyl CoA, butyryl CoA, and butyryl glutathione were prepared by the anhydride method as described in METHODS IN ENZYMOLOGY (Stadtman, 1957) as based on an earlier procedure (Simon and Shemin. 1953). Complete reaction of SH groups was ascertained with nitroprusside reagent (Stadtman, 1957) and if necessary for completion of the reaction additional anhydride was added. The reactions were carried out at 0°C and were monitered with a pH meter. The pH was maintained at 6 or above by addi- tion of 1 N KOH and when reaction was complete the pH was 27 adjusted to 6. For quantitative determination of thiolester the hydroxamic acid was formed and color deveIOped by addi- tion of ferric chloride. Succinic anhydride afforded the basis of the standard curve even though the color values are slightly different from acid to acid. The original procedure (Stadtman, 1957) was scaled down to 0.30-ml total volume in order to increase sensitivity and the umoles assayed are equal to 0.9 times net A540 mu' Lactyl CoA was prepared by the mixed-anhydride reaction between ethylchloroformate and free lactic acid and subsequent diSplacement of ethylformate by COASH (Flavin, 1963). Inasmuch as the reaction mixture con- tains large amounts of tetrahydrofuran and pyridine, mixed-anhydride thiolester preparations were always sub- Jected to continuous liquid-liquid extraction with diethylether at 0°C. Lactyl CoA was found to be very labile to storage and purification. Column chromatography on Bio-Gel P-2 at about 1°C resulted in complete hydrolysis of thiol- ester. Lactyl thiolesters are surprisingly unstable to storage at -14°C, see Table 1. After 8 days only 14% of the original thiolester remains. When stored at pH 8 they are even more labile as 100% hydrolysis occurs with- in 3 days. Chromatography of lactyl CoA and lactyl-§y panthetheine on Sephadex G-15 (1 x 50 cm column, flow 4 ml/hr) was the only successful purification step. The 28 Table 1. Lability of lactyl thiolester to storage AMOUNT OF THIOLESTEB COMPOUND AMOUNT OF initial after 8 days HYDROLXSIS at pH 6, -14°C Butyryl-§r glutathione 19 umole/ml 15 umole/ml 21% Lactyl-fig glutathione 21 3 81 29 yields were nearly quantitative if the fractions were con- centrated as a liquid on a test tube Botovap; however, lyophillization resulted in complete hydrolysis. The column was operated as quickly as possible and the temper- ature was that of the cold room, about 5°C. Lactyl CoA is probably labile due to the neighboring effect of the ElEEE' hydroxy group. This could occur in at least two ways. First the inductive effect: a hydroxyl is electron-with- drawing and would thus promote attack by water on the car- bonyl carbon of the ester: HD’OH | 0 T -C-¢¢ @ - 6 H20’///a Second, the hydroxyl might be acting as a nucleophile and diaplacing the thiolester. alpha-Hydroxycarboxylic acids are very prone to lactone formation (Fieser and Fieser, 1956). However the lability must be more complicated or else lactyl CoA would be stable to lyOphillization which was not found to be the case either by Rabin g£_§l. (1965) or in these studies. Many bimolecular reactions have recently been shown to occur much faster in frozen solu- tion than in liquid water at the same temperature, e.g., the mutarotation of glucose (Kiovsky and Pincock, i966) and the solvolysis of ethylene chlorohydrin (Pincock and Kiovsky, 1966). The eXplanation seems to be that there is 30 a concentration effect, i.e., the reactants are localized in small micelles of liquid water within the ice lattice and the effective concentration of reactants increases dramatically. Acrylyl CoA, or acrylyl-§gpantetheine, must be pre- pared with precaution to prevent addition of excess CoASH across the double bond of acrylyl CoA. This problem is minimized by employing the mixed-anhydride method with inverse addition, i.e., a small amount of COASH solution is slowly added to a large excess of acrylyl-mixed-anhyd- ride at 0°C. Purity of acrylyl thiolester is best measured by taking a difference Spectrum in such a way as to give a spectrum of the thiolester alone with no contribution from CoA or other chromOphores such as the common contam- inate, pyridine, which also absorbs in the 260 mu region. The procedure is to take two identical aliquots of the acrylyl thiolester, and to hydrolyze #1 in 0.1 N_KOH (% hr, room temp) and then to neutralize by addition of an equivalent amount of HCl. To aliquot #2 one adds equivag lent amounts of KOH and HCl simultaneously. The two ali- quots are diluted to identical volumes and scanned in a SpectrOphotometer employing matched cuvettes. Aliquot #1 is placed in the reference beam and #2 in the sample beam. A Cary 15 recording Spectrophotometer was used for these determinations. I am grateful to Professor Karl Decker for his suggestion of this procedure. 31 Acrylyl CoA and other acrylyl thiolesters are stable to storage. However purification proved to be impossible. The thiolester was completely hydrolyzed by passage through Sephadex G-15, DEAR-cellulose, Dowex-i (HCOS), Dowex-i (thSphate, pH 6.25), and ECTEOLA-cellu- lose columns. On other columns, (Dowex-50(H+), CM- Sephadex, and CM-cellulose), the thiolester linkage remained intact, but extensive polymerization occurred as determined by difference Spectra and actual precipi- tation (turbidity). Such lability is expected as acrylic acid itself polymerizes Spontaneously unless stabilizers are added (Feairheller and Katon, 1967). Acrylyl CoA can be made‘in‘gigu just prior to its use by pre-incubation of buffer, acetyl CoA, acrylate, and CoA transferase. Consistent with the hydrolysis of acrylyl thiolesters during passage through columns with basic functional groups such as DEAE- is that amine buffers are known to catalyze thiolester hydrolysis (Koch and Jaenicke, 1962). The Situation is complicated by acid-catalyzed cationic polymerization of acrylyl CoA on acidic columns, e.g., CM-Sephadex. alpggfiPhOSpholactic acid was prepared as the barium salt by (1) reaction of ethyl lactate with polyphOSphoric acid, (2) selective saponification of the ethyl moiety, and (3) alcohol precipitation of the salt as described previously (Cherbuliez and Rabinowitz, 1956; Cherbuliez and Rabinowitz, 1959). Lactic acid cannot be phOSphory- 32 lated directly on account of pronounced dehydration and lack of desired product. PhOSpholactate is somewhat dif- ficult to hydrolyze. Its half-life in acid-molybdate solution is 46 min at 100°C compared to 24 min for 2,3- phOSphoglyceric acid (Rose and Pizer, 1968). Calcula- tions based on the known acid strengths of monoesters of phOSphoric acid (Kumler and Eiler, 1943) Show that phos- pholactate should have pKa's about 1.5, 3.5, and 6.5. These figures correlate well with those for 2—phOSpho- glyceric acid which are known to be 1.8, 3.63, and 6.64 (Ballou and Fisher, 1954). Acetyl phOSphate was prepared from dipotassium hydrogen phOSphate and acetic anhydride and isolated as the dilithium salt as described in METHODS IN ENZYMOLOGY (Stadtman, 1957) according to the procedure of Avison (1955). 3-(§gGlutathionyl)propionyl hydroxamate was pre- pared by the mixed-anhydride method employing a large excess of glutathione and acrylyl mixed-anhydride (Flavin, 1963) to form 3-(§rglutathionyl)prOpionyl glutathione; subsequently, the hydroxamate was formed from the thiol- ester by adding neutralized hydroxylamine. In this instance, advantage is taken of the reactivity of acrylyl thiolester, i.e., the excess glutathione adds to the double bond as soon as acrylyl glutathione is formed. As described in the RESULTS Section, this reactivity provides a means of trapping acrylyl CoA. 33 Diaphorase, phOSphotransacetylasse, and CoA trans- ferase were prepared and assayed as described previously (Baldwin 23 al., 1965). The optical assay for CoA trans- ferase with butyryl CoA as substrate (Barker 32.21., 1955) proved to be satisfactory for qualitative determinations such as those employed for purification. 2-Keto-3-deoxy- 6-phOSphogluconate aldolase (KDPG aldolase) was the gift of my colleague, L. R. Barran. Acrylyl CoA aminase was prepared by rupture of Clostridium prOpionicum cells grown on beta-alanine. bgtggAlanine induces the enzyme to levels 200 times those of cells grown on alpha-alanine and a higher Specific activity is difficult to attain (Vagelos g2.gl., 1959); hence the very active extracts were used without purification. ASSAIS FOR THE INTERCONVERSION OF LACTATE AND ACRYLATE Throughout the course of this work, a unit is defined to conform with IUPAC recommendations, i.e., 1 unit of enzyme forms 1 umole of product per minute at a temperature which should be Specified (Dixon and Webb, 1964). .Assays were performed at room temperature or at 370 as indicated. Protein determinations were performed in triplicate by both the Warburg-Christian and the Waddell methods as described in TECHNIQUES IN PROTEIN CHEMISTRY. The methods usually agree, and the average value was taken. 34 (1) COUpled.Assay The assay for lactyl CoA dehydrase was assembled from partially purified components as described (Baldwin §£_gl,, 1965) and was later modified to consist of the following: (1) 0.08 M phOSphate buffer, pH 7.5; (2) 0.80 mg.2gpggg-iodOpheny1-3-pgggynitrophenyl-5-pheny1tetra- zolium chloride (INT); (3) 0.01% gelatin; (4) 0.06? MM phenazine methosulfate (PMS); (5) 13 mM_NAD; (6) ca. 0.004% yeast alcohol dehydrogenase (ADH) (1.5 units); (7) 1 mM acrylyl CoA. The modified coupled assay is inherently superior to the original one because none of its components is obtained from g, elsdenii. Yeast ADH recognizes lactyl esters, but not free lactate, as an alcohol: evidently the enzyme has little steric Specifi- city for that portion of the substrate molecule but does require it to be uncharged. The molar extinction coeffi- cient for the formazan produced from INT upon reduction was taken as 14,200 liter per mole-centimeter at 490 mu (Hirsch g; 2;” 1963). INT is the dye of choice because its formazan is fairly stable to oxygen and is more sol- uble than others, though gelatin is still desirable to prevent precipitation (Nachlas 33 21.. 1960). During validation of the assay pyruvyl dinitrOphenylhydrazone was formed and isolated according to the procedure of Neish (1957). 35 (2) Direct Assay All attempts at measuring lactyl CoA dehydrase activity by observing the appearance or disappearance of acrylyl CoA Spectrophotometrically at 263 mu were unfruit- ful. The variations attempted were (1) 0.06 M triethanol- amine-HCl, pH 7.5: 6 mM_acrylyl pantetheine: 0.35 mg of enzyme protein; and water to 0.21 ml (Vagelos g§_§;., 1959), (2) the same as above with NAD, LDH, and CoA trans- ferase added individually or in combination and with acrylyl CoA as substrate, (3) 0.05 M_phOSphate, pH 7.5, or 0.05 M_HEPES, pH 7.5, mM EDTA, mM DTT, HM Fe (II) and all combinations thereof with acrylyl CoA and acrylyl pantetheine as substrates, and (4) 0.05 M_perphOSphate buffer, pH 8.5, with purified lactyl CoA as substrate. Furthermore the addition reaction, e.g., of 5 mM DTT and 8.75 mM_acrylyl pantetheine in 0.10 M triethanolamine-HCl buffer, pH 7.5, was easily and reproducibly observable; thus the acrylyl thiolesters were present in the assay mixture, and the SpectrOphotometer, which has the advan- tage of a linear reSponse to an absorbance of three, is capable of making the necessary measurements. (3) Manometric Assay At pH 7.5 or less, extracts form equimolar amounts of H2 gas and propionate from lactate. Hence measurement of the H2 evolved in a Warburg apparatus affords an assay 36 for the lactate to propionate reactions among which is that catalyzed by "lactyl CoA dehydrase." The activity values obtained from this assay are minimal inasmuch as other enzymes in the pathway may be limiting. The incubations were carried out as described by Ladd and Walker (1959), except that 1 mM DTT was added. Hereafter this will be referred to as incubation system 1. The reactions were stopped after 45 min by addition of an equal volume of 0.1 H.3230H- Other incubations were carried out as follows: (1) nitrogen atmOSphere; (2) room temperature; (3) vari- able substrate: (4) 0.10 M phOSphate buffer (pH 7.0); (5) 5 m ATP: (6) 5 mill P155012; (7) 25 L121. acetyl CoA: (8) 0.10 mM.NAD; (9) 1.0 mM DTT; and (10) extract. These are essentially the conditions of Evans and Jackson (1966), except DTT was added and dialysis of the extract was normally omitted. These conditions will be referred to as incubation system 2. Distribution of nitrogen to many tubes was facilitated by a needle-valve, aquarium manifold which was purchased from a local pet shop. (4) Propionate Assay Gas chromatography of prOpionate which is formed from lactate is a valid assay if CoA transferase and acyl CoA dehydrogeanse are not rat limiting: CoA-T lactate + acetyl CoA selectyl CoA + acetate 37 -H20 lactyl CoA > acrylyl CoA rds + ACD acrylyl CoA + 2e“ + 2H ::prOpionyl CoA CoA-T V prOpionyl CoA + acetate acetyl CoA + propionate. NET: lactate + 2e" + 2H+——————)propionate + H20. Incubations were carried out under N2 and at 37°C; the assay solution consisted of 0.033 M_TriS'HCl (pH 7.6); 0.33 M_sodium.DL71actate (pH 7.6); variable ATP as Speci- fied; 5 mM MgC12; 25 “E acetyl CoA: variable NAD(H) as Specified; and extract. NADH can be added as a source of electrons for the reduction of acrylate; however, since this assay was only employed with crude extracts the lactate dehydrogenase(s) maintained sufficient levels of reducing equivalents as to make addition of NADH unneces- sary. This assay suffers from being dependent on many enzymes for activity and such dependency precludes its use for purification of enzyme activity. The initial idea was that as purification was attempted the enzymes necessary for reduction of acrylyl CoA would separate and then acrylate would appear as the product. Acrylate forms a unique peak on GLC and can be taken as an index of activity. However deSpite numerous purification attempts this assay, as described above, never gave acrylate as product. 38 Propionate and other acids were determined quanti- tatively by gas-liquid chromatography (GLC). The appara- tus was a dual column Packard Instrument Co. gas chromato- graph equipped with dual hydrogen flame detectors and dual-pen Texas Instrument Co. recorder. The 2 mm-diameter columns were packed with 10% FFAP on Chromosorb W, acid washed DMCS, 80/100, which is produced by Wilkens Instru- ment Co. The temperatures used were column 120, inlet 145, outlet 190, and detector 155°C. The sensitivity of the system permits determination of 1 nmole of any volatile fatty acid. 50 ul samples of the incubation solution were withdrawn at regular intervals and mixed with 10 ul 1 M 32804 and 50 ul 0.0105 M_Sodium isobutyrate which served as an internal standard and eliminated errors due to injection. The peaks were integrated manually by multi- plying height by half-height width. (5) Acrylate Assay Acrylate was formed as the major product when extracts were added to a solution to give 0.30 ml total volume and the following concentrations of components: (1) 0.033 .11 HEPES buffer (pH 7.75): (2) 7 mM MgC12: (3) 0.02% methylene blue; (4) 0.3 mM acetyl CoA; (5) 33 mMHQL-lactate (10 umoles), and (6) 33 mM acetyl phOSphate. These assays were performed under N2 and at 37°C. Acrylate was determined on samples taken at various times and 39 injected into the gas chromatograph in the same manner as propionate. LACTYL CoA DEHYDRASE PURIFICATION When the coupled assay was thought to be valid, lactyl CoA dehydrase was purified according to published procedures (Baldwin 23 21., 1965; Baldwin, 1967), except that the calcium phOSphate gel step was omitted for lack of reproducibility. The partially purified enzyme obtained in this way was applied to various columns in an attempt to achieve additional purification. All column chroma- tography was done at about 5°C. After addition of sample, a Sephadex G-100 column (1 x 73 cm) was eluted with 0'01.fl Tris-H01 buffer, pH 8.5. The eluent was passed through a flow cuvette which was kept at about 5°C and was monitered at 280 and 260 mu with a DU Spectrophotometer equipped with Gilford automatic cuvette and wavelength positioners (Wood and Gilford, 1961). A Bio-Gel P-150 column (2.5 x 50 cm) was poured and Operated in similar fashion, except that 5(10)-4 M_DTT and 5(10)-4 M sodium acrylate were added to the eluting buffer. DEAR-cellulose was washed and poured into a column (1.2 x 50 cm). Prior to application, the sample was dia- 2” and 10"3 11 BAL which is a redox lyzed against 10"6 MlFe buffer (Wood, 1967). The eluent consisted of a linear gradient of increasing ionic strength with 1 mM_DTT throughout: distilled water-———+solution of 0.2 M phOSphate 40 and 0.4 M sodium chloride (pH 7.5). Hydroxylapatite was prepared as described in TECHNIQUES IN PROTEIN CHEMISTRY (Bailey, 1962). The gel was poured into a 3.4 x 45 cm column to a height of 40 cm. Undialyzed enzyme was added and eluted step-wise with increasing concentrations of phOSphate buffer pH 6.8. OTHER ENZYME ASSAYS Acyl CoA dehydrogenase was assayed as prescribed by Baldwin and Milligan (1964) with the exception that prOpionyl CoA was substituted for butyryl CoA as sub- strate. QeLactate dehydrogenase was assayed Spectrophoto- metrically by following the disappearance of ferricyanide at 420 mu upon reduction by lactate. The procedure used was originally that described by Symons and Burgoyne (1967) but was modified to eliminate perphOSphate and EDTA when a metal requirement was found and consists of mixing the following, in order, 200 pl of 0.1 M_Tris (HCl) (pH 8.0) and 0.1 M.leactate (sodium salt); 50 pl of 0.1 fl K3Fe(CH)6: 15 ul of 20 liCoClZ; and 35 ul of extract or water. The extinction of ferricyanide was taken as 1,040 liter per mole-centimeter relative to ferrocyanide. The existence of pyruvate produced during the course of an Optical assay was correlated with activity by forming the dinitrophenylhydrazone at various times according to the direct method of Friedemann and Haugen (1943), i.e., 41 strong alkalai was added to the pyruvyldinitrophenylhydra- zone solution (to give 0.5 N NaOH overall) in order to decompose excess dinitrophenylhydrazine and to dissolve precipitated proteins; and the absorbance was measured at 435 mu without organic-solvent extraction. ‘L-Lactate dehydrogenase activity was determined in a similar manner except that L-lactate was substituted for Delectate as substrate. Lactate racemase activity was measured by allowing racemase activity to convert Delactate to L- lactate and the latter was removed by an excess of rabbit muscle L-lactate dehydrogenase, and either NAD at pH 9.9 according to the procedure of Lowry (1957), or 3-acetyl- pyridine NAD (AchAD) at pH 8.5 as described by Dennis and Kaplan (1960). The advantage of the NAD analogue is that it affords a linear assay in the direction of pyru- vate formation because the lactate + AchAD to pyruvate + AchADH equilibrium is shifted to the right relative to NAD. The extinction coefficient of AchADH relative to AchAD is 7,750 liter per mole-centimeter (Kaplan and Ciotti, 1957). Two kinase assays were employed. The first mea- sures ATP consumption Spectrophotometrically in an indi- rect manner. ATP is provided by PEP, ADP, and pyruvate kinase; as ATP is consumed so is PEP which releases pyruvate; pyruvate appearance is coupled to NADH with rabbit muscle LDH and observed at 340 mu (Anderson and Wood, 1967). The second depends on disappearance of 42 acetyl phOSphate as measured by hydroxamate formation, e.g., if acetyl phOSphate and lactate react to form ace- tate and phOSpholactate and Since phOSpholactate gives a negative hydroxamate test, then the reaction can be followed as described. The procedure was to mix in order 100 pl of 0.1 H HEPES buffer (pH 7.75): 35 ul of 60 my; MgClZ: 6 ul of 1% methylene blue; 30 ul of 1 mM dinitro- phenol; 42 ul of 1 M acetyl phOSphate; 42 ul of 1 M sodium lactate; 30 ul of 5 mM acetyl CoA; and 50 ul of water or extract. The assay was incubated at 37°C under N2, and at various times aliquots were removed for hydrox- amate formation as described above for thiolester deter- mination. ORGANIC ACID PURIFICATIONS Propionate and acetate were purified by chromato- graphy on either a Wiseman-Irvin or Swim-Krampitz column as Specified (Wiseman and Irwin, 1957) (Swim and Krampitz, 1954). The latter column was modified as described by Kuratomi and Stadtman (1966). The packing of these columns was greatly facilitated by use of a tamper (30 x 1/8 in stainless-steel rod with 7/16 in diameter, perfor- ated, stainless-steel disc on the end). Since these columns do not separate acrylate and propionate, acrylate was, in Specified cases, removed by acidifying the solu- tion to 0.4 NpHBr and heating in boiling water for 2 min. GLC analysis showed the acrylate to have disappeared. 43 Both cationic polymerization and hydrobromination probably occur. Propionic acid was converted to acetic acid by Schmidt degradation and permanganate oxidation according to the procedure of Phares (i951) employing the apparatus described by Krichevsky and Wood (1961). Lactic and pyruvic acids were purified by chroma— tography on Celite columns according to the procedure of Swim and Krampitz (1954) as modified by Kuratomi and Stadtman (1966). Lactate was determined in two ways. First, in the Barker and Summerson procedure (1957). lac- tate was oxidized to acetaldehyde which subsequently reacts with‘p-hydroxyldiphenyl to form a colored adduct with absorbance at 570 mu. Lactate was also determined by enZymatic conversion to pyruvate as described above for the lactate racemase assay. The enzymatic determin- ation is the superior method of the two, eSpecially if 3-acetyl-pyridine NAD is employed, because of the ease and reproducibility with which it is performed. In either case a standard curve was run with each set of determinations. PhOSpholactic acid was purified for purposes of determining radioactivity by descending paper chromatog- raphy on Whatman 3 MM paper with 3:1 95% ethanol-0.1 N acetate buffer (pH 4.0). Several different solvent sys- tems were tested for ability to separate phOSpholactate, 44 phOSphate, and lactate from one another with the following results: PhOSpho- , Solvent RF: PhOSphate. lactate Lactate n-Propanol:HC00H:H20 (6:3:1) 0.49 0.51 0.78 HCOOH:H 0:95% EtOH (1829878) 01+? 073 078 2-Butanone:HOAc:H20 (8:8:1) .59 streak 1.00 95% EtOH:0.1 Ii Phthalate pH 3.0 (3:1) 2.6 .63 .68 95% EtOH:Dioxane:H20:H0Ac (60:20:19:1) streak .50 .70 95% EtOH:0.1 N Acetate pH 3.0 (3:1) .26 .63 .68 95% EtOH:0.1 N Acetate pH 4.0 (3:1) .25 .40 .60 95% EtOH:0.1 NDAcetate pH 508 (331) 057 035 075 Further purification was accomplished by placing the phos- pholactate on a DEAE-cellulose column (1 x 40 cm) equilib- rated with 0.03 M_ammonium carbonate and eluting with a linear gradient consisting of 200 ml of 0.03 M ammonium carbonate to 200 ml of 0.03 M ammonium bicarbonate at a flow rate of 30 ml/hr. Three ml fractions were collected. This column effects very clean separation of phOSpholac- tate from lactate. The remaining contaminant was phos- phate and was difficult to remove. Specific precipita- tion with triethylamine-molybdate of orthOphOSphate is supposed to be possible (Sugino and Miyoshi, 1964); 45 however all attempts in this regard were unsuccessful because the phOSpholactate either was destroyed or was precipitated along with the phOSphate. The best separa- tion of phOSpholactate and phOSphate was achieved on a Sephadex G-10 column (2.5 x 100 cm) operated at 10 ml/hr, though a second passage was necessary to achieve quanti- tative separation. PhOSpholactate was determined either by radioactivity measurements or by alkaline phOSphatase treatment followed by determination of the lactate and orthOphOSphate released. Alkaline phOSphatase treatment was performed at pH 8 in the presence of 5 mM_MgC12. PhOSphate was determined by the method Of Chen gfi,gl. (1956) and, in order to increase sensitivity, the determinations were performed on a 0.80 ml basis instead of 8.0. betaeAlanine was determined by TLC chromatography on pre-coated Silica Gel plates in 80:20:4 Methanol: WaterxPyridine as described by Brenner gt 2;. (1965). The Spots were visualized by Spraying with ninhydrin and heating for 10 min at 110°C. In this system beta-alanine has BF 3 0.44 compared to 0.58 for alpha-alanine. RADIOACTIVITY MEASUREMENTS Radioactivity was measured in a Packard Tricarb Liquid Scintillometer. For counting tritium and carbon- 14 the settings were tritium channel gain 50, window 50- 400, and carbon-14 channel gain 10, window 200-1000. For 46 double-labeling experiments the tritium counts should be about ten times those of carbon-14 because 36.7% of the channel B counts also appear in channel A. Bray's scin- tillation fluid was used while working with tritium because it works well for samples containing large amounts of water (Bray, 1960). Chemiluminescence is somewhat troublesome; hence the samples should be thoroughly cooled and then counted twice. For counting carbon-14 and phos- phorous—32 the settings were carbon-14 channel gain 9.0, window 50-600, and phOSphorous-32 channel gain 1.2, window 200-1000. XDC scintillation fluid (xylene, dioxane, cellosolve) was used for all work other than that involv- ing tritium (Bruno and Christian, 1961). MASS SPECTROMETRY L—Lactate-Z-IBO was prepared by exchange between H2180 and pyruvate as catalyzed by KDPG-aldolase and by subsequent reduction with NADH as catalyzed by muscle lactate dehydrogenase (Rose and O'Connell, 1967). The lactate was purified by chromatography on a Celite column 180 content of the 2-180-lactate as described above . The was determined by converting it to carbon dioxide as catalyzed by mercuric chloride at 500°C according to the procedure of Rittenberg and Ponticorvo (1956); the C02 was then analyzed on a mass Spectrometer. 18O-Lactate was converted to propionate and 18O-phOSphate by reaction with extracts in incubation system 1 (see Manometric H7 Assay). The phOSphate was isolated and purified as described by Boyer and Bryan (1967). The 18O of phos- phate was converted to 18O-carbon dioxide by heating with guanidine-HCl according to the procedure of Boyer 9:2 2;. (1961). Carbon dioxide samples were analyzed on a low resolution mass Spectrometer by the Department of Chem- istry, Michigan State University. CHEMICALS Most of the chemicals used in the course of this work are listed below according to their sources. Corn steep liquor was a gift of the A. E. Staley Manufacturing Co., Decatur, Illinois. CHEMICAL COMMERCIAL SOURCE antimycin A Sigma Chemical Co. 1,10-phenanthroline°H20 " ‘ oligomycin " glucose-6-phOSphate " dinitrOphenol " LOU-lactic acid " pyridoxal°HCl " sodium pyruvate " yeast alcohol dehydrogenase " muscle lactate dehydrogenase " rotenone " trisodium PEP " sodium acrylate K and K Rare Chem. Co. al ha-amine red-R " dichTorophenolindOphenol, sodium salt " ethyl chloroformate " hydracrylic acid " INT dye " sodium azide " coenzyme A (lithium salt) P-L Biochemicals, Inc. ATP, ADP, and.AMP " 48 CHEMICAL NAD and NADH NADP glucose-6-phOSphate dehydrogenase glutathione, reduced CTP 3-acetylpyridine NAD cysteine°HCl dithiothreitol D-lactic acid, lithium salt 2,2'-dipyridyl Sephadex G-10 Sephadex G-15 Sephadex G-100 CM-Sephadex C-50 60% sodium lactate isobutyric acid isovaleric acid acetic anhydride butyric anhydride succinic anhydride succinic acid Bio-Gel P-2 Bio-Gel P-150 Dowex-i Dowex-50 DEAR-cellulose CM-cellulose ECTEOLA-cellulose sodium arsenate potassium cyanide potassium_ferricyanide sodium acetate potassium phOSphate, monobasic QLrlactic acid sodium azide sodium pyrophosphate phOSphoglycolic acid, cyclohexylammonium salt disodium EDTA-ZHZO oxalic acid-ZHZO COMMERCIAL SOURCE P-L Biochemicals, Inc. It I! I! II II Calbiochem w I! I! Pharmacia Fine Chem., Inc. II fl " PfaHStlehl Lab. . InCO Eastman Organic Chemicals n I! I! I! H Bio-Rad Labs‘ II '9 I! II n N Mallinckrodt 0 n I! I! It Fisher Sci. Co. I! General Biochemicals J. T. Baker Chem. Co. 49 CHEMICAL COMMERCIA1.SOURCE sodium arsenite Matheson, Coleman, and Bell methylene blue Eberbach intestinal alkaline phOSphatase Worthington. RADIOCHEMICALS Lactic acid-2-14C, lactic acid-3-14C, and tritiated water (1 C/gm) were purchased from Volk Radiochemical Co. Acrylic acid-i-lu C was obtained from International Chemical and Nuclear Corp. (ICN). Adenosine-S'-triphOSphateegamm_- 32? was obtained from The Radiochemical Center. SodiumeD- lactate-lac (u) and sodium-Qgplactate-i-luc were from Amersham/Searle. Lactic acid-3-3H was prepared by taking advantage of the KDPG aldolase-catalyzed exchange of the hydrogens of pyruvate (Meloche and Wood, 1964b). The following rea- gents were added and stirred (1) 0.50 ml of water; (2) 0.050 ml of tritiated water; (3) 0.500 ml of 0.05 M phos- phate buffer (pH 7.5): (4) 110 mg of sodium pyruvate (one millimole); and (5) 0.010 ml of KDPG aldolase (47,000 U/ml). The solution was allowed to incubate at room tem- perature for three hours to assure complete exchange. Then pyruvate was reduced to lactate by adding rapidly a second solution which was prepared by mixing in order the following (1) 3 ml of 0.05 M_phosphate buffer (pH 7.5); (2) 10 mg of NADP: (3) 364 mg of glucose-6-phOSphate; (4) 0.100 ml of muscle lactate dehydrogenase crystals; 50 and (5) 0.050 ml of glucose-6-phOSphate dehydrogenase crystals (suSpended in ammonium sulfate solution). The combined solution was permitted to incubate at room tem- perature for sixteen hours. The solution was evaporated to dryness on a test tube Rotovap and the lactic acid was purified on a Swim and Krampitz column. The yield was 72% and the lactic acid-3—3H had a specific activity of about 0.18 uC/umole. It was stored as a 30 mm solution (pH 7) at -14°C. CHAPTER IV RESULTS The eXperiments presented herein are divided into two sections: (1) those which consider the difficulties of obtaining a lactyl CoA dehydrase assay and the subse- quent reinvestigation of acrylyl CoA as an intermediate and (2) those which show a new c-phOSpholactyl CoA inter- mediate between lactyl CoA and acrylyl CoA. The first section will involve: (a) validation of the coupled assay; (b) validation of the modified coupled assay; (c) partial purification and prOperties of lactyl CoA dehydrase; (d) the inconsistencies of the lactyl CoA dehydrase which involve Specific activities, inhibitors, and the lack of a direct assay; and (e) further evidence for acrylyl CoA as an intermediate using new types of; experiments. The second section will include: (a) the propionate assay and its stimulation by ATP; (b) stabil- ization of extracts as regards their activity in the assay for propionate formation; (c) purification of the prOpionate-assay activities; (d) inhibition by dinitro- phenol and its reversal by ATP; (e) demonstration of 180 transfer from 2-180-lactate to orthOphOSphate concomitant with propionate formation; (f) labeling Of-phOSpholactate from.ggmm_-32P-ATP; (g) the assay for acrylate formation 51 52 and its requirement for acetyl phOSphate; (h) the enzyma- tic formation of phOSpholactate from inc-lactate and 32P- orthophOSphate; (i) isolation of the intermediate (phos- pholactate); (j) the conversion of enZymatically- and chemically-synthesized phOSpholactate to acrylate; and (k) chemical characterization of phOSpholactate. I. DIFFICULTIES WITH THE LACTYL CoA DEHYDRASE ASSAY AND REINVESTIGATION OF ACRYLYL CoA AS AN INTERMEDIATE The starting point of this investigation was the assembly of the components of the coupled assay for lactyl CoA dehydrase according to the procedure of Baldwin (1962). The coupled assay was subjected to validation tests as described below. A. Confirmation of Lactyl CoA Dehydrase Activity The coupled assay assumes ready reversibility of the lactyl CoA-acrylyl CoA interconversion and involves the lactyl CoA dehydrase-catalyzed conversion of acrylyl CoA to lactyl CoA which is observed Spectrophotometrically by coupling the formation of lactyl CoA to the reduction of a tetrazolium dye (INT): 1) acrylyl CoA + H20_____4.lactyl CoA CoA transferase 2) lactyl CoA + acrylate + INT p coupling system pyruvate + acrylyl CoA + INT reduced' 53 The assay was subjected to the following validation tests: (1) the appearance of pyruvate concomitant with the reduc- tion of INT was demonstrated by isolating pyruvyl dinitro- phenylhydrazone, and (2) the discrimination of the coupling system against crotonase activity was confirmed by its failure to oxidize betaghydroxypropionate. 1. Eight different coupled assay mixtures contain- ing lactyl CoA dehydrase, which had been subjected to protamine sulfate treatment and one ammonium sulfate pre- cipitation, were pooled and allowed to react an additional hour. The mixture was incubated with 2,4-dinitrophenyl- hydrazine reagent and a sample was Spotted for chromatog- raphy. The paper chromatogram was run in descending fashion with 7:1:2 nebutanol-ethanol-0.5 M ammonia. The Spots correSponding to those of authentic pyruvyldinitro- phenylhydrazone were visible following incubation but were absent at zero time. This experiment, though admittedly gross, suggests that pyruvate was formed from acrylyl CoA. 2. The following components of the coupled assay system were mixed with beta-hydroxyprOpionate or lactate, and the absorbance at 485 mu was recorded: (1) buffer, (2) INT, (3) gelatin, (4) NAD, (5) PMS, and (6) lactate dehydrogenase. The beta-hydroxypropionate solution was prepared by neutralizing 0.10 ml of beta-hydroxypropionic acid and diluting to 1.0 ml. The sodium lactate solution was prepared by diluting 0.10 ml of 60% sodium lactate syrup to 1.0 ml. The data obtained (Table 2) showed that 54 Table 2. Specificity of coupled assay for conversion of acrylyl CoA to lactyl CoA SUBSTRATE AMOUNT ADDED 4A/5 MIN umoles None -- 0.145 bgtg-Hydroxypropionate 6.5 0.145 " 13.0 0.060 ” 19.5 0.040 Lactate 6.5 3.30 " 13.0 10.0 " p 19.5 ' 10.0 Each reaction cuvette contained in 0.250 ml: 0.04 M pyrophOSphate buffer (pH 8.5); 0.80 mM INT; 0.01% gelatin; 0.067 mM_PMS; 13 mM_NAD; substrate; and about 1 unit muscle lactate dehydrogenase. 55 rabbit muscle lactate dehydrogenase did not utilize beta- hydroxypropionate and hence is Specific for the alpha- hydroxy group under these conditions. Therefore the coupled assay is not a measure of crotonase activity. B. The Modified Coupled Assay for Lactyl CoA Dehydrase The coupling system of Baldwin's original coupled assay consists of three enzymes, of which CoA transferase and diaphorase were prepared from g. elsdenii: CoA transferase 1) lactyl CoA + acrylate s;lactate + acrylyl CoA LeLDH + 2) lactate + NAD+ > pyruvate + NADH + H diaphorase 3) NADH + H+ + INT : NAD+ + INTreduced. Although not described here the Baldwin assay system was restudied in an effort to eliminate the use of enzymes from.g, elsdenii and this resulted in the following modi- fications (for details see MATERIALS AND METHODS). First the CoA transferase and muscle lactate dehydrogenase were replaced by yeast alcohol dehydrogenase (ADH) because it had been reported by Rabin g£_§l. (1965) that lactyl CoA was oxidized directly by alcohol dehydrogenase whereas lactate was not. Second the diaphorase was replaced by phenazine methosulfate (PMS): ADH 1) lactyl CoA + NAD+ a; pyruvyl CoA + NADH + H+ 56 PMS 2) NADH + H+ + INT a NAD+ + INTreduced. The modified coupled assay was tested in two ways. First the ability of the coupling system to measure lactyl CoA was compared to that of the original coupling system wherein lactyl CoA dehydrase itself was not of concern but rather whether the product of its action on acrylyl CoA, lactyl CoA, could be rapidly oxidized. Consequently lactyl CoA was added directly. Equivalent amounts of purified lactyl CoA were added to each assay system and the total change in A485 was observed (Table 3). The modified system gave slightly lower values, probably because the lactyl CoA was partially hydrolyzed. In a second test, equivalent amounts of lactyl CoA dehydrase were added*to the original and modified assay Systems in order to compare activities. The initial rates were about the same in both cases (Table 4). The modified coupled assay is inherently superior to the original for the following reasons: (1) it elim- inates any components which are derived from.£, elsdenii other than lactyl CoA dehydrase; (2) whereas CoA trans- ferase is required in the Baldwin assay, the modified assay makes use of alcohol dehydrogenase to oxidize lactyl CoA directly making CoA transferase unnecessary; and (3) since alcohol dehydrogenase is Specific for the lactyl portion only, whereas CoA transferase requires CoA esters, it would be possible to use or at least to test the 57 Table 3. Validation of the modified coupled assay using alcohol dehydrogenase and purified lactyl CoA ASSAY TOTAL CHANGE IN A485 Experiment 1 2 Original 0.75 1.43 Modified 0.60 1.00 Each reaction cuvette contained in 0.250 ml: 0.04 M perphOSphate buffer (pH 8. 5); 0. 80 mM INT; 0.01% gelatIn; 13 mM NAD, and about 15 OM lactyl CoA. In addi- tion the original assay contained about 1 unit of muscle LDH and 0.1 unit of diaphorase; the modified assay con- tained 0. 067 mM PMS and 0.004% yeast ADH. 58 Table 4. Comparison of the coupled assays for lactyl CoA dehydrase ASSAY EXPERIMENT ORIGINAL MODIFIED units per ml Extract 1 0.0792 0.0477 2 .981 .259 Partially purified fraction 2 .150 ' .082 3 .200 .218 The assays were performed as described in Table 3 except 1 mM acrylyl CoA replaced lactyl CoA as substrate and lactyl CoA dehydrase was added. One unit of enzyme is defined as that which forms 1.0 umole of product per minute at room temperature (about 28°C inside the Spec- trophotometer). 59 pantetheine thiolesters. Both coupled assays have a com- mon shortcoming: dithiolthreitol is required by the dehydrase but it also Spontaneously reduces INT thereby giving rise to high blanks. C. Partial Purification and Properties of Lactyl CoA Degydrase Employing Baldwin's procedure (1962; Baldwin §£_§;,. 1965), without the final step, the average of ten purifi- cations yielded a Specific activity of 0.107 umole/min/mg protein with a standard deviation of 0.114. This repre- sents an average purification of 20-fold. The highest activity was 0.625 or 109-fold. Further purification was attempted using Sephadex G-100, Bio-Gel P-150, and DEAE-cellulose. In every case all activity was lost. Recombination of various fractions or addition of boiled enzyme supernatant did not recover the activity. Also no evidence for dissociation of a cofactor was obtained by charcoal treatment of the par- tially purified enzyme. The enzyme, as measured by the coupled assay, does require an SH reducing agent, eSpecially during storage. Extracts were prepared in the absence of dithiothreitol (DTT), assayed, and then stored at 5°C. After 43 days, the activity was 15% of the original value and addition of 1 mM DTT resulted in over 100% return of activity (Table 5). 60 Table 5. Dependence of the coupled assay and of the sta- bility of lactyl CoA dehydrase upon dithiothre- itol TIME DTT CONCENTRATION ACTIVITY Days mM' units per ml 0 0 0.111 43 0 0.014 43 1.0 0.148 The assay mixture contained 0. 08 M Tris-acetate buffer (pH 8. 0); 0.80 mM INT; 0. 01% gelatin; 13 mM NAD; 1 mM acrylyl CoA; 1 unit of muscle lactate dehydrogenase; about 0.1 unit of diaphorase; and lactyl CoA dehydrase in a final volume of 0.250 ml. One unit is the same as 111. Table ’4'. ‘ 61 Up to this point, most of what Baldwin discovered about lactyl CoA dehydrase had been confirmed. However, several difficulties had already presented themselves as discussed below. D. Evidence Against a Simple Lactyl CoA Dehydrase The troublesome aSpects of the coupled assay were: (a) the failure to demonstrate either the disappearance of acrylyl CoA or the production of acrylyl CoA from lactyl CoA using the direct SpectrOphotometric assay for the acrylyl thiolester bond; and (b) the extremely low Specific activities measured by the coupled assay in g, elsdenii. Also the behavior of the partially purified lactyl CoA dehydrase was unusual. Additional purifica- tion attempts, or in some cases storage, resulted in com- plete loss of activity. Recombination of fractions which might be necessary if a cofactor had been separated was not successful. Most dehydrases fall into one of four groups: (1) no cofactor requiring, (2) divalent metal ion requiring, (3) metal ion and reducing agent requiring, and (4) pyridoxal phOSphate requiring (Malstrbm, 1961). Lactyl CoA dehydrase requires a reducing agent, but metal ion cofactors are eliminated inasmuch as EDTA is not an inhibitor and is sometimes beneficial. Pyridoxal phOSphate or cobalamin (vitamin B12) does not appear to be required because charcoal-treatment had no effect on 62 activity, and their function would not be eXpected on mechanistic grounds. On account of the above difficulties the very existence of lactyl CoA dehydrase was reexamined as discussed below. The existence of lactyl CoA dehydrase was tested in three ways: (1) Ladd and Walker had Shown that 2 x 10"5 M cyanide and other inhibitors of electron transport phOSphorylation prevent the conversion of lactate to acrylate as well as the reverse reaction. If it is pre- sumed that these inhibit by acting on the "lactyl CoA dehydrase" then the coupled assay should be inhibited to the same extent. (2) The overall rate of prOpionate for- mation Should be less than or equal to the rate of lactyl CoA dehydration. Hence, if the Specific activity of lactyl CoA dehydrase is less than that for the overall reaction, either it cannot be considered as an obligatory enzyme of the acrylate pathway, or the dehydrase assay is defective relative to the fermentation mixture forming propionate. (3) The direct Spectrophotometric assay for the appearance or disappearance of acrylyl CoA must be possible with a "lactyl CoA dehydrase" and so a variety of assay conditions were employed in an effort to find one which gave enzyme activity. (1) The coupled assay was used to measure lactyl CoA dehydrase activity in unpurified extracts in the presence of varying concentrations of sodium cyanide, sodium azide, sodium ethylene diamine tetraacetate (EDTA), 63 and hydroxylamine hydrochloride. AS shown in Table 6, the concentrations necessary to affect a 50% inhibition are much higher than those reported for the interconver- sion of lactate and acrylate under similar conditions as measured by evolution of hydrogen in the Warburg apparatus (Ladd and Walker, 1965). The manometric assay is valid at pH 7.5 or less because extracts form equimolar amounts of H2 gas and propionate from lactate. Complete inhibi- tion of the coupled assay was difficult to observe because the reSponse to inhibitor was not linear at high concentra- tions. The failure to observe inhibition at concentrations as low as those used by Ladd and Walker may be interpreted to mean that the lactyl CoA dehydrase activity observed in the coupled assay is not related to the enzymes of the acrylate pathway. (2) The Specific activity of fresh extracts as measured by the coupled assay was reexamined. Thirteen different fermentations, assayed with the original coupled assay system, gave an average Specific activity of 0.00578 umole/min/mg protein with a standard deviation of 0.00772. Calculation of the minimum Specific activity possible assuming that (a) there is log phase growth with no lag, and (b) the enzyme activity increases gradually throughout growth, doubling every generation, gives 0.684 umole/min/mg protein (for details see APPENDIX, p. 1). The discrepancy between calculated and observed Specific activities is greater than loo-fold. 64 Table 6. Inhibitors of lactyl CoA dehydrase _ CONCENTRATION NECESSARY FOR f INHIBITOR 50% INHIBITION OF 100% INHIBITION OF , COUPLED ASSAY WARBURG ASSAY* HE mH Cyanide 2.2 0.02 Azide 8.0 2.0 EDTA 12 - Hydroxylamine 35 0.1 *Data of Ladd and Walker (1965). The coupled assays were performed as described in Table 5. 65 (3) The direct assay for lactyl CoA dehydrase depends on the appearance or disappearance of acrylyl CoA as observed Spectrophotometrically at 263 mu. gipgg,gg£§- Unsaturated thiolesters have a unique absorbance peak at 263 mu with a molar extinction coefficient of 6,700. The assay was attempted under the following conditions: (1) acrylyl pantetheine as substrate in Tris buffer (pH 7.5) with NAD, LDH, and CoA transferase added individually and in combination, (2) acrylyl CoA as substrate with the above variations, (3) acrylyl CoA or acrylyl pantetheine as substrate in phosphate or HEPES buffer, 1 mM EDTA, 1 mM DTT, 1 0M Fe (II), and all combinations thereof, and (4) lactyl CoA (purified) as substrate in pyrophosphate buffer (pH 8.5). The addition reaction between dithio- threitol and, for example, acrylyl pantetheine was easily and reproducibly observable; thus the acrylyl thiolester was present and observable. Yet in every case there was no enzyme catalyzed appearance or disappearance of acrylyl CoA in the direct assay whether extracts or partially- purified lactyl CoA dehydrase was used. The amount of enzyme assayed from varied from 0.01 to 100 times that required to give average rates in the coupled assay. With these disturbing facts in mind, it was con- cluded that the lactyl CoA dehydrase as defined by Baldwin and confirmed in this study is some artifact or Side reac- tion and does not represent the reaction which is the object of the present study. Thus, a new approach was in 66 order. Before continuing with the purification and study of the key enzyme of the pathway, the following questions ought to be answered with certainty. (1) Is acrylate an intermediate or is the reaction via a totally different mechanism, e.g., hydride diSplacement as in deoxycytidine diphOSphate formation? (2) What are the individual reac- tions of the pathway, i.e., does the lactyl CoA to propionyl CoA conversion involve one, two, or three separate reactions? (3) How are the enzymes of the indi- vidual steps assayed? AS a first step, the existence of acrylyl CoA as an intermediate was reexamined. E.- Confirmation of AcryMyl CoA Intermediate (1) Larsson showed that when deoxyribonucleotides are formed from ribonucleotides in tritiated water, tritium is not incorporated into the 3' position of ribose. The results were interpreted as evidence of a hydride dis- placement of the 2' hydroxyl group. This sort of experi- ment depends on equilibration of the potential hydride hydrogen with the tritiated water. In the case of deoxy- ribonucleotide formation such was known to be the case. It is likely that in propionyl CoA formation from acrylyl CoA the reducing hydrogens would equilibrate with the sol- vent Since acyl CoA dehydrogenase either utilizes a cyto- chrome component, in which case the hydrogens would be derived from protons of the solvent, or a flavin component 67 in which case the reducing hydrogens would exchange with those of the solvent; on the other hand, prOpionate for— mation by hydride diSplacement seemed possible by analogy with deoxyribonucleotide formation. 'This possibility was tested by: (1) incubating lactate and extracts in tri- tiated water; (2) isolating the propionate produced; and (3) determining the relative amount of tritium incorpor- ated into positions 2 and 3 of propionate. If hydride displacement of the hydroxyl group occurs, tritium would not be incorporated at carbon 3; and, if elimination of the elements of water occurs, acrylyl CoA would be an intermediate and tritium would be found at carbons 2 and 3. Specifically, lactate-Z-luc or lactate-3-1uc (120 umoles; 5.3 x 108 cpm/mole) was incubated in system 1 (see MATERIALS AND METHODS) with tritiated water (1.2 x 1010 cpm). The propionic acid produced after 45 min was purified by partition chromatography, assayed for radio- activity, and degraded to acetic acid. The acetic acid was purified as above and counted. The percent of the total tritium incorporated into carbon 3 was calculated from the following equation: %3H in 0-3 .-. counts 3H in AC . counts 3H in PRO counts C in AC counts C in PRO 14 The C affords an internal reference, and hence exact titrations and quantitative transfers are not necessary. The isotope content showed that about 26% of the tritium 68 incorporated into propionic acid is in the C-3 position (Table 7) and the remainder is in the C-2 position. Also if one assumes that two tritium atoms Should be incorporated per propionate, then the eXpected 3H/luc ratio would be 302; the observed ratio was 60 (see APPENDIX, p. 3, for the details of this calculation). Thus, there is an isotope discrimination of about 5-fo1d. This is well within the range usually encountered (Melander, 1960). Since considerable tritium was incorporated at carbon 3 a direct hydride diSplacement of the hydroxyl group is eliminated as the sole mechanism and the data are consistent with an acrylyl intermediate. Otherwise no tritium would have been found in C—3 as was observed in the deoxycytidine diphOSphate case by Larsson (1965) and by Durham.ggwgl. (1967). The data are also consis- tent with the supposition that the reducing protons for the acyl CoA dehydrogenase reaction equilibrate well with the solvent. The fact that tritium is not distrib- uted equally between carbons 2 and 3 may be due to slight differences in isotope effect, i.e., the acyl CoA dehydro- genase may discriminate less at carbon 2 than at carbon 3. The difference may also be due to a loss of some tritium at carbon 3 during the conversion of propionate to ace- tate inasmuch as the Schmidt degradation and permanganate oxidation impose severe conditions. 69 sz mqummawm .mmomemz ma confluence mm doomawoo cam oopmaoma mos beehom cpmsoaaoaa one .SHE m: Hon 2 Home: comm as was soapmwsOsd one .Ha m.~lmo mafiaob Hmpop a ma Aaao Hoa H N.Hv Hops: oopmapHHWIwmm ”BBQ :8 H.o “moo Hmpooc as H.o ”Aoaoa\sao oa N m .USaim no INVIOpopomHTAQ z m:o.o ”Anaopoaa we Ha pzonmv poohpwo «Ao.o may Meagan opmsamosa z mo.o ”wsazoaaom on» dosaopsoo manpwaa coapomoa 0:9 “5.:auv n.0N Au.:HHv m.mm ommaobd am on sm.m sam.mm me~.aoa m.ma Hom.mm mmo.aom n ma am mm.~ oom.am o:m.am m.aa Hao.oa mmm.emm m n so mmm.o mma.mem Som.mm mo.s mso.mm meo.mom H mm mm ma.~ oso.mm mma.maa :a.m mem.ma «ma.wm m am an am.a Hmm.mo~ sma.mom sm.m oao.oa mam.esm N an ac mom.H ana.mm~ rmm.omm mm.m ebm.om sos.sam a Aora\mmv mum Nlo 0H9 m CH ohdeHa soapommh one mpmsodmona op mpmpoma wcapnmbsoo mmamNCm on» no mpaaanmpm no owmhopm mo mm mo pommhm .N onswdm In moégm . N m m . # _1N_Qo % IllvaO ode . w . .mvoo ogqo. Ail/\IIOV 3.3.93.3 ’ 86 adding Ca012 and K3P04 to the extract as described by Ochoa (1955). The proportions were modified so that 0.12 volume of 15% CaClz solution was added to the éxtract and 0.2b volume of 10% K3P°u solution was added to the result- ing supernatant. The final supernatant after removal of the gel by centrifugation was called calcium phOSphate_ gel "supernatant" fraction, and the eluate of the first precipitate obtained with 0.3 fl'phOSphate buffer (pH 7) was called calcium phOSphate gel "eluate" fraction. Both fractions were necessary for production of propionate though each, and eSpecially the second, had residual activity (Table 13). It was concluded that two enzymes had been partially separated. To decide which fraction acted on lactate, presumably to produce an intermediate utilized by the other fraction, the fractions were incu- bated individually for 1 hr at 37°C under N2, then the solution was immersed in boiling water for 20 sec. Fin- ally the other fraction was added to the incubation and samples were taken at various times. As shown in Table 1h, fraction 1 followed by 2 produced prOpionate whereas the reverse order did not. It was thus concluded that fraction 1 acts on lactate to produce an intermediate used by fraction 2. The intermediates possible were lactyl CoA, phos- pholactyl CoA, acrylyl CoA, or some new and unknown com- pound. Lactyl CoA was eliminated because both fractions .are more active with reSpect to CoA transferase than they 87 Table 13. The activity of calcium phOSphate gel frac- tions in converting lactate to prOpionate % ORIGINAL.ACTIVITY EXPERIMENT ELUATE AND SUPERNATANT SUPERNATANT 1 68% 20 2 63 17 3 86 -- 4 26 i6 5 0* -- 6 0* -- 7 13 0 8 5 0 9 17 0 10 6 0 11 100 0 12 30 #7 13 8 -- in 51 12 15 94 208 16 57 25 average 39 22 *PMSF omitted (see APPENDIX, p. 3 and Appendix Figure 1). Assay reaction mixture consisted of the following: 0.022 M Tris buffer (pH 7.6); 0.022 M MM-lactate; 0'022.§ ATP: 0.65 mM’Mg012; 0.16 mM acetyl CoA: 20 ul of each frac- tion: and water to give 93 ul total volume. Incubation was at 37°C under N . Aliquots were withdrawn at intervals for gas chromatographic analysis as described in MATERIALS AND METHODS. The eluate alone usually has less than 1% of the original activity. 88 Table 14. Determination of order of function of calcium phOSphate gel eluate and supernatant fractions 2; OF ORIGINAL ACTIVITY ORDER OF FUNCTION EXPERIMENT 1 2 3 Eluate then Supernatant 89 256 20 Supernatant then Eluate 7 0 9 The assay reaction mixture consisted of the follow- ing: 0.022 M Tris buffer (pH 7.6); 0.022 M DL-lactate; 0.022 M ATP: 0.65 mM MgCl ; 0.16 mM acetyl 03K; 20 ul of each fraction: and water go give 93 pl total volume. Incubation was at 37°C under N2. Aliquots were removed at intervals for gas chromatographic analysis as described in MATERIALS.AND METHODS. 89 are for the lactate-to-propionate conversion. Also acrylyl CoA was eliminated because (1) acrylate did not accumulate when fraction 1 and lactate were incubated in the propion- ate assay system and (2) acyl CoA dehydrogenase activity in both fractions was greater than the rate of the overall system forming propionate. At this point the earlier observed occasional.ATP stimulation and the separation into two fractions by cal- cium phosphate gel fractionation made the case for a phos- pholactyl intermediate extremely promising. The next step seemed to be the development of a lactyl CoA kinase assay and its subsequent purification. However the high.ATPase activity of extracts precluded demonstrating a lactyl CoA dependent disappearance of ATP (cf. MATERIALS AND METHODS, general kinase assay). The ATPase rate was 6_umole/min/mg protein or at least 10 times that of the lactate-to-pro- pionate enzymes. Furthermore addition of lactyl CoA slowed rather than increased the loss of ATP. Without an assay for the presumed kinase, purification was not a practical means of demonstrating the reactions involved in the conversion of lactate to propionate and was tem- porarily abandoned. C. Indirect Evidence for PhOSpholactyl Intermediate ’ Before this work was undertaken Ladd and Walker (1965) had demonstrated that active phOSphate compounds 90 stimulate the interconversion of lactate and acrylate in dialyzed extracts of g. elsdenii. Furthermore the inter- conversion was inhibited by uncouplers of oxidative phos- phorylation. At first their findings were puzzling but as the difficulties with the "lactyl CoA dehydrase" deveIOped they took on new meaning and were reconsidered. (1) Dinitrophenol (DNP, 10'”’M) inhibits 100% the conversion of lactate to propionate by fresh extracts (Ladd and Walker, 1965). There is a slight increase in the levels of prOpionate during an assay with DNP but it is at the eXpense of endogenous acrylate. ATP is able to reverse the inhibition of DNP; both the rate and extent of propionate formation increases with increasing amounts of ATP. The effect of ATP on the extent of propionate formation was demonstrated by dialyzing extracts for 4 hr against 0.05 M phOSphate buffer (pH 6.5) and by incubating the dialyzed extracts in the prOpionate system with vary- ing levels of ATP for 24 hr to insure complete reaction; the result is that about 2 moles of product are formed per mole of ATP added (Figure 3). The value is consistent with the existence of phOSpholactyl intermediate if one assumes that the stoichiometry is 3 lactate-———————9 2 acrylate + 1 acetate, because the acetate forms 1 ATP or in otherwords the phos- phoroclastic system gives 1 ATP per 2 moles of product. This 1 plus the 1 added gives 2 moles of ATP per 2 moles or product, 1030 o 91 .803: Qz< mumHmmsdz ma bondhommd mm mammamnm oasamamOpmaohso new no“ Um>oamh one: mmaaamm as :N nopmd .Nz nouns manpmnmaamp 8009 pm was doameSOGH .madaob Hapop HE oo.m Op Amps; Una “HonmSQOHpazac_m :Ioa “Ada Hog Camachm mg HC possum so as o.m ”a: 63636» “BS as H £92 as 20 3.8 38¢ a: mu “mama: as m ”mpwpoflumm m 8.0 “84. m3 $.83 3.36693 w. 8.0 ”wedsoa Iaom map confidence chapxaa soapomma 029 .H: : Umuhamdu mama mpomhpwm mas an Hamnmbmh “coapmahom mpmzoanonm ho soapanasna HonoSQOHpHnHQ .m mnsmam 9.2 mm VN $3023 884 E4 ON m_ N. m , Honoomn. m0 mMJOE m._ a._.< “.0 MAO—2 mun. .\ .\. Q “- i\. 0 44mmm>mm . n_._.< do ._.Z.m._.xm uZO_._._m_T_Z_ QZO O O N ..__ 0 r0 . (8310M) . HIV-113W CINV EiVNOIdOHd 93 1 ATP 1 ADP 3 lactate 2 phOSpholactate 1 ADP ' 2H 1 pyruvate 1) 2H 2 acrylate or propionate . w 1 acetate + 1 ATP Oligomycin inhibited prOpionate formation and again ATP reversed the effect (Table 15): the fact that reversal is not complete may be due to (1) ATP not being the direct phOSphoryl donor or (2) an essential cofactor having been removed during dialysis. (2) 180 Transfer from 2-180-lactate to orthophos- phate. In order to further implicate phOSpholactate as an intermediate an eXperiment was designed to determine if 180 of lactate-2-180 would be converted to orthOphos- phate-180. As noted previously in phOSphoryl transfer P-O cleavage occurs whereas in elimination C-O cleavage would be eXpected: ° fi -SCoA g clz-SCoA X~P 318003 a @O-P-18O-CH ('23 \x e". ' 3 EH3 0 0 T 18 ll Go-I'u OH + f-SCOA 0 CH 6) ll 0 n: K) 94 Table 15. Inhibition of propionate formation and its reversal by ATP , INHIBITOR CONCENTRATION ATP ADDED ACTIVITY M, umoles units None 0 0.0042 " 5 .0330 n 15 .7000 Oligomycin 10‘” 0 0.0 " " 5 .0041 n n 15 .0139 Dinitrophenol 10'4 0 0.0 " " 5 ' .0020 " n 15 .0050 The reaction mixture consisted of the following: 0. 06 M phOSphate buffer (pH 7. 0): 0. 01 M DL-lactate: 5 mM Mg012 3 5 uM acetyl CoA: 0.10 mM NAD; variable ATP as indicated: variable inhibitor as indicated: 2. 0 ml of extract (39 mg protein/ml); and water to 5.0 ml. The reaction was incubated at 37°C under N2. At intervals samples were removed for gas chromatographic analysis as described in MATERIALS AND METHODS. 95 O-lactate synthesized had an atom % excess of 18 18 0 The 2- = 1.16%. Since it was formed by equilibrating pyruvate with 5.58% 18O-HZO the value eXpected was 1.86; thus dur- ing the reduction of pyruvate to lactate some 180 was lost. The 2-180—lactate was converted to propionate by incubation with extracts according to the conditions of 18O of the propionate assay. If it is assumed that the lactate is transferred to phOSphate every time prOpionate is formed, the eXpected atom % excess of 18O in phOSphate can be calculated: eXp. = (1.16 at. % ex. in lac)(3 O in lac)(umole PRO formed) (4 O in P1) (umole P1 in system) The data show excellent agreement between eXpected and observed values of 18O in phosphate in some cases. The greater discrepancy in the other values is attributable to the small amount of sample analyzed for 18O and to the oversight of not accurately determining the inorganic phOSphate present. However in every case the data sug- gest at least partial transfer of 180 from lactate to phOSphate (Table 16). (3) gagggrBZP-ATP Labeling of the intermediate. Demonstration of the transient accumulation of intermedi- ate was undertaken using 32P-labeled.ATP (gamga—labeled) and.Q£r1actate-1u0. Thus the proposed intermediate should be doubly labeled. Specifically 0.25 mC of gamm_- l4 32P-ATP and 1 uC of Qgelactate- C (u) were incubated 96 Table 16. 180 Transfer from lactate to phOSphate con- comitant with propionate formation ATOM % EXCESS 18O IN CO2 FROM PHOSPHATE EXPERIMENT EXPECTED FOUND 1 0.266 0.0397 2 5.22 0.249 3 5022 5.45 4 5.22 3.48 The incubation mixture consisted of: 0.06 M 1midazole buffer (pH 7.0); 5 mM ATP; 5 mM MgC12; 25 DM acetyl CoA: 0.10 mM NAD; 1 mM DTT; 2.6 mM M—lactate-Zz180; 2.0 m1 of extract (31 mg/ml); and water to 5 ml. Incu- bation was at room temperature for 150 min. The ortho- phOSphate was isolated and equilibrated with C02 for analysis as described in MATERIALS AND METHODS. 97 with extracts in the "propionate assay? incubation system. After 30 min the reaction was stopped by addition of 5 volume of ethanol, and the denatured proteins were removed by centrifugation. A small portion of the supernatant was spotted on Whatman 3 MM paper and developed by descending chromatography in 95% ethanol:dioxane:water:acetic acid (60:20:19:1). The levels of ATP were so high that counts were distributed over the entire chromatogram. Therefore the remainder of the incubation mixture (about 4.0 ml) was treated with 50 mg of activated charcoal which had been previously shown to effectively remove £222_-32P- ATP. The resulting supernatant was chromatographed as described above. The radioactivity was located by cutting the strip into pieces each representing deltaeRF Spans of 0.0625. Chemically synthesized phOSpholactate was shown to have an RF of 0.5 in this system. A 32F labeled peak was found at HP = 0.5: further there was a slight shoulder at R 1” 0.5 in the C-lactate peak at RF = 0.57 (Figure F _ 4). Though the chromatogram suggested the formation of phospholactate, the evidence was still weak because the lac-lactate had not been separated and because the radio- active-phosphate peak streaked so broadly that the 32P- phOSpholactate peak consisted of but one point. (4) The acrylate assay. A system for conversion of lactate to Only acrylate (or acrylyl CoA) was desirable because (a) it would eliminate the requirement for acyl CoA dehydrogenase and lactate dehydrogeanse activities 98 .Aaumauomuowv Uaom odpmomuhmpwz “Ocmxoapuaozmspm Rmm ma asamhmopmaopno mzaccmommo an umaoambmu USO Hogan 22 m smapmsz op UOHHQQO was maaamm 02p 20apmwsmanpcmo med pcmapdmhp amounmso hmpm4 .Hocm:pm mo madaob m mo soapdpvm mp Umaaopm was Scapomom .caa om Mom Nz hound comm ad was :oHpmn:onH .Ha o.m op amps: Uzm “AHE Hem sampOHQ ma Hmv pomhpxm .8 as a; neaoEpfifim .6833 ya a “mach: a 8.0 “manuammulstmm ca 3.0 new a: a 8.0 :3 oaaumpaeomaumm on a new engofiufl a 8.0 “84. m3 .823 mpmnamosa a 00.0 “mmdzoaaom exp no Cowmamcoo maszaa coapmnsOma one mpwcoaaosm op mumpoma mo soamambsoo 05p ca mpmacmshopna cm on mewlmmm1> aoam mmm mo nmmmzmha .: mhswam no... 86 who one hCiao one 9.0 009m 086 coon I‘m..- . ooo.o_ w ._ W 538255. . omnmm< 1‘:: (r reduced dye propionyl CoA acetate The oxidation Of lactate by NAD-independent dehydrogen- ase(s) is the primary source of the reducing electrons (see APPENDIX, p. 4) and probably involves flavins. Thus dyes capable of accepting electrons from flavoproteins such as methylene blue and phenazine methosulfate are likely acceptors (Nachlas 22 §;., 1960). The acceptors tested were 2§p___-iod0pheny1-3-aw-nitrophenyl-S- phenyltetrazolium chloride (INT), PMS-ascorbic acid, methylene blue, dichlorophenoindophenol (DCPIP), and ferricyanide. In most cases both acrylate and propionate accumulated when 25 umoles of the acceptor was added to 101 the assay (Figure 5). Inasmuch as methylene blue was by far the most effective acceptor (see also Table 17), various amounts were added to the assay in order to Optimize its effect. The optimum concentration is 0.02% or 0.160 umole per assay (Figure 6). However the Speci- fic activity with respect to acrylate accumulation was lower than that obtained from the usual propionate assay. Therefore, in an effort to achieve a reSpectable rate of acrylate formation, a series of phOSphoryl donors were screened. Also, in order to dramatize the effect and thereby identify the primary phOSphoryl donor, the extracts were dialyzed for 5 hr against 0.05 M phosphate buffer (pH 6.5) and 1 mM DTT prior to testing. In this manner it was found that acetyl phOSphate is by far the most effective donor in stimulating acrylate accumula- tion (Figure 7), and when added the Specific activity is higher than that of the usual propionate assay without methylene blue (0.272 umoles/min/mg vs. about 0.1). Besides methylene blue and acetyl phOSphate, the other requirements of the acrylate assay were expected to be magnesium ion and, by analogy with propionate assay, catalytic amounts of CoA thiolester. At first a magnesium ion requirement was not observable: however, for this and other reasons the assay was modified to contain 10 umoles of lactate instead of 100 (assay volume 0.300 ml). With this assay as described in MATERIALS AND METHODS under "acrylate assay," the magnesium 102 .mammamzm monasvm pmmOH ache madam nomad “caom cannoomdlmzm Amy .aHaoo Asa .eeaceaoaaaea Ana .BZHumzm ANA .eaan ecoaanpm: AHV .mmomemz 92¢ mAdedez :« Umndhcmmc mm mammadzd oasamhwomeOHno new you cmboamn Ohms mOHaamm mambhmpnd .N #4 z Mecca comm as was :oHpmnSonH .Oasaob Hmpop HE oom.o mbaw on Hopes use “Rafi pea campona we mcv pomnpwm no H1 om “Hopamoom no macs: mm ”460 Hapeoe_wa mm.o ”ma<.ma m.o “maowz_ma a nepeeeeaxmm_m mm.o ”Aw.m mac Hemmdp maaa_m mmo.o ”wzazoaaoc on» mo cmpwamsoo mOHSQNHa hmmmd neonaooom nonpomam msoanmb mo mmmcmbapoommm “mumpoma Bosh noapmafiazoom mpmahhod .m mhsmdm mMHDZS (SETOWH) HIV—MHOV we an on em m. ..N_ _ m .. “w , . . . _ at. on m . on om on mmoEmoo< zoEomd can? E3284 104 Table 17. Rate of acrylate accumulation from lactate: . effectiveness of various acceptors _ ELECTRON ACCEPTOR ACRYLATE.ACCUMULATION E8 umole/min volts Methylene blue .089 0.011 PMS—INT -.O11 -- Ferricyanide .008 -.360 DCPIP .028 .217 PMSqucorbic acid .015 .080 *Eé Values from RESPIRATORY ENZYMES, 1949. Assays were performed as described in Figure 5. 105 .mmcmemz cad quHmmadz :a condhomoc we mammamnm oasaethpmaonso mew Mom cmboaoh Ohms medaadm .madbnmpaa 94 .NZ House comm us one: m20apdflfionH .Oadaob Hence H1 com mbaw on Hope: new ”Se 8a 5893 we m3 peaapee .8 H1 03 ”as... as Rm .48 H2366... as 86 «~85 ma Tm 3:3 magenta 633a? ”manpoflflm a mmé “3.x. mac acumen mama z mmo.o ”wCHEOHHOh on» no cmpmfimcoo mehfipxaa memmd coapehpzeonoo mean OzOHmSpOa co uommmm ”mumpoma Bonn scapmflsasOom opdamnod .m Onswam 106 ”.540 mzmfizems. “e. 000 00.0 00.0 00.0 9.0 9.0 00.0 0 o\o mmod 20.20.5828 moEmooq .10 0602 ESEQA BiVWAHOV lm’. W . O «00.0 1 . 3 S / $000.0 .W. N / W .0000 e . ..0 no 1000.0 m . B . W 0.0.0 chmmm 107 .mmomamz 924 quHmmadz :« confluence me opmamaod pom samhwopManso new on» so emumamse use mambhmpsa pm emboamn one: moaaamm .Nz nouns comm no one: onadeSosH .Oasaob Hence an oom obaw op amps: use "Ada Hon adoponn me No ”as m commamaev pomppwm mo H1 0: "doc Hmpoom_ma Nd.o «Hococ Hanonamona a 20.0 :33 623.203 RN00 ”~83 ma 0.m ”opmpoaauwm a 000.0 234. may Mommas mammm_fl mmo.o ”weazoaaom on» no copmamzoo mohdpxaa hmmmd mpdoamaooam nosoc Hmnosamona «evapoma Bonn soapmasasooa opmamhod .m Chewam mmEbZS mm em 0? mm IIO azq N n54 n_._.< an mzozll 108 £0 a Daemoq N (I) q- (\l A ._ (salowfi) 31V1A80V~ A Om $5200 #mozamoza .10 1 mmmzm>fi ommmm 109 ion and thiolester requirements were pronounced (Table 18 and Figure 8). The characteristics Of acrylate formation are cer- tainly consistent with involvement of phospholactyl inter- mediate. Nonetheless the following alternative was con- sidered. PhOSphoenolpyruvate (PEP) was tested as an intermediate by adding it alone as substrate to the assay. The specific activity obtained in this manner was but 9% of that on Mgrlactate without any phOSphoryl donor. Acrylate formation from PEP is probably due to hydrolysis and subsequent reaction of the pyruvate to form acetyl phosphate and lactate which react to form phospholactyl CoA and then acrylate. PhOSphoglycollate was tested as an analogue of phOSpholactate. There was an absence of any new peak on the gas chromatograph which would have indicated its utilization; further when lactate was added to the assay, its conversion to acrylate was inhibited by phOSphoglycol- late. D. Direct Demonstration of a New Intermediate (Presumably a-PhosEholactzl CoA) Up to this point the phospholactyl intermediate hypothesis had been established in several indirect experiments. However a direct demonstration of the appearance of the intermediate and of its conversion to acrylate was now desirable. In the eXperiments described 110 Table 18. Acrylate assay requirements DELETION SPECIFIC ACTIVITY umole/min/mg protein None 0.036 Lactate 0.007 MgC12 0.042 Acetyl CoA 0.006 Acetyl phOSphate 0.015 Methylene blue 0.020 Assays were performed as described in Figure 7 ?xcept that acetyl phosphate was the phOSphoryl donor 0.033 M_. 111 .OHSpHaa henna OS» on emcee mm: AHS\stpOHQ we mmv pd no an 0: use .coumaeae no: we: pomnpwm on» pas» pamoxm m Onswum ed confluence mm coshomhmn one: whemmd Nacwz you unmaonaswmn ”mpmpoma song scandanom mamahnod. .m shaman x250 20.252828 N.00.). S N_ o. 0 0 - e x N . 0 M0.0.0.. ...ll..o_.0 *, m . - 0.0 , as m e - . . .\ 00.0. e. . . _ HZMEMEDOMK 44.52 .>mo< Ail/010v 0|:ll03d8 113 below it is shown that (1) a transient intermediate appears during the course Of an incubation, (2) if the intermediate is isolated, purified, and added to another incubation mixture, it is converted presumably via phos- pholactyl CoA to acrylate, (3) chemically synthesized phospholactate forms acrylate similarly, and (4) the intermediate is characterized to show its identity as phOSpholactate. 14C_ (1) Experiments showing the appearance of a and 32P-labeled intermediate. (a) The formation of an intermediate was demonstrated by incubating extracts in the "acrylate assay" with M-lactate-lnc (u) and 32P- orthOphOSphate. In all probability the 32P-phosphate is converted to acetyl phosphate: lac-lactate —. lactyl CoA 1"Cu-32P-lsbelec1 intermediate 1 pyruvate 32 P1 32P—acetyl phosphate Following a 60 min incubation, enzymes were inactivated and removed by ethanol precipitation: and, after centri- fugation. the entire supernatant was Spotted on a TLC plate and developed with (60:20:19xi) 95% ethanol: dioxane:water:acetic acid. A.1uC- and 32P-labeled spot was formed with an RF value of 0.12; chemically synthe- sized phospholactate diSplayed an RF value of 0.13. 114 However the spot overlapped that of orthOphOSphate (RF = 0.03). The incubation and TLC-chromatography was repeated, and then, following drying, a second chromatog- raphy was run in formic acid:water:95% ethanol (1:29:70) to the point where the second front was 0.44 as far as the first. The plate was scored into gggtggRF = 0.0526 sections; the silica gel coating was scraped into scin- tillation vials, one section per vial. Plots of inc and 32P content versus RF showed a single double-labeled spot (Figure 9). The 140 in the double-labeled peak represents 12% of the total label added as lactate-140 and the ratio of 32P/lh'C indicates that there are about 0.7 phosphate/ lactyl moiety. However the lack of a labeled orthOphOS- phate peak probably means that the second solvent system also failed to resolve the intermediate and phOSphate. In an effort to find an appropriate solvent system, studies were conducted with chemically synthesized phos- pholactate prepared as described in MATERIALS AND METHODS. The best system proved to be 3:1 95% ethanol:0.1 MDacetate buffer (pH 4). Employing this system for chromatography of incubations prepared as above, the separation from orthOphOSphate was better but still not complete: the RF- value of orthOphosphate is 0.25 compared to about 0.4 for phOSpholactate. Nevertheless, by plotting changes in radioactivity as a function of time (compared to zero f 14 time) vs. RF in this system the appearance o C- and 32P-labeled intermediate was evident (RF 2 0.38) (Figure 10). 115 .Opeduoehopea uOHOneHIOanou 0:» ed opepoea\openamo£a m.o peeps mad when» pass mummwwsm cea\m~m mueSOO no cause one .ede on you «2 House comm as we: scapensOeH .Oesaob Hmpop H1 com ebaw on have: use «Ade Hen sampOHn we mcv avenues mo H1 om “doc Hmpood_me m.~ “epdsnmosmospHOImNm c1 «.0 beans :3 03-30833. 00 01 3.0 seeps ”epssosanfl m 3.0 20;. m3 seamen mdha.m mao.o .weaxoadom esp mo uopmameoo massage soapoeon esa mopmapmnem me opmsnmosaOspHOIm use mpdpodfl cha wadedmpeoo onspwae sodpoeon co asaewwopmeo o Homealeana .m chewam ISOO I200 <3- 62. L) 600 300 RADEOACTIViTY 0 hi TLC PLATES I I500 SECOND , 5 E * FRONT , I LACTATE \\8 f 1 1200 DOUBLE- - ACRYLATE = LABELED ; 0.. 900_COMPOUND__ 900 S a 3%. g 6000 . 1 500 J5 .30 105 115 .Oumdumehoued ueHOanIOHnsou on» ed opmpoeH\openamosm m.o psond one when» peep mpmowwsm c:H\mNm muesoo mo oHpmH 0:9 .eHe ow you Nz nouns comm pm we: GOHmesOeH .OeSHob Hana» H1 oom obaw on Hope: use aaHe Hon sampOHA we mwv poenpwo no H1 om “doc theoe_ae m.m ”endgamonaoanOImNm 01 a0 Ross :3 03-33874 so 01 3.0 snobs ”sesaosaumm m. 3.0 :04. may acumen mHHB.fl mHo.o .weHxOHHou on» no uopmameoo waspwde eOHpomOH one mendemeSm me opmSAmonaospHOIm use mumpomH :c:H weHeHmpeoo ohepwae eoHpomOn mo asaeawopmeo o HommHlsana .m OstHm |500 I200 <3- 62. L) 600 300 RADEOACTIViTY 0 hi TLC PLATES I I500 SECOND , 5 E * FRONT , I LACTATE \\8 f 1 1200 DOUBLE- - ACRYLATE = LABELED ; 0.. 900_COMPOUND__ 900 S a 3%. g 6000 . 1 500 J5 .30 105 117 Figure 10. Separation of 11‘C-. and 32P-labeled inter- mediate The reaction mixture consisted of the fol- lowing: 0.033 M,HEPES buffer (pH 7.75): about 0.01 uC of 3gP-orthophosphate; 0.033 M_QM-lactate; about 0.07 00 of Marlactate-i-luC; 7 mM MgCl2: 0.02% methylene blue: 0.5 mM acetyl 09A; 20 ul of extract (53 mg protein per ml): and water to give 150 ul total volume. Incubation was at 37°C under N2. .After deproteinization the samples were analyzed by descending paper chromatography in 3:1 95% ethanol:0.1 M_acetate buffer (pH 4); phOSpholactate standard HF e 0.39. 14C A CPIVI 32p A CPIVI 1 1 El, ANALYSIS OF L‘AIELING 0N PAPER . In I \ - ACRYLATE I500 /———\——— , I I “ 0 MIN ’ IOOO 6 \3‘8 ——‘— 500 I I 900 ‘ I! I g; 60 IVIIN I II/ ‘ I \ I 0 MIN 600—-— ~—-—-——I~\I—’ .____i___ I I I I I I \ I‘ I \\ i_._ __.__1____ _____________.___.___.. 300 ‘ I \ I' I l/\ I I .\ O 3.. ,1, l mum—wremrw moraines: ”Tm—35m O 0.2 0.4 0.6 0.8 117 Figure 10. Separation of 140- and 32P-labeled inter- mediate The reaction mixture consisted Of the fol- lowing: 0.033 M,HEPES buffer (pH 7.75): about 0.01 00 of 32P-orthophOSphate: 0.033 M QMPlactate; about 0.07 00 of Mgrlactate-i-luC: 7 mM MgClZ; 0.02% methylene blue: 0.5 mM acetyl 09A; 20 ul of extract (53 mg protein per m1): and water to give 150 01 total volume. Incubation was at 37°C under N2. After deproteinization the samples were analyzed by descending paper chromatography in 3:1 95% ethanol:0.1 M'acetate buffer (pH 4); phOSpholactate standard RF 2 0.39. 14C A CPIVI 32p A CPIVI 1 1 El, ANALYSIS OF LAIELING 0N PAPER . In I \ - ACRYLATE I500 /———\——— , I I “ 0 MIN I IOOO 6 \3‘8 ——‘— 500 I I 900 ‘ I! I g; 60 IVIIN I II/ ‘ I \ I 0 MIN 600—-— ~—-—-——I~II—’ .____i___ I I I I I I \ I‘ I \\ i_._ __.__1____ _____________.___.___.. 300 ‘ I \ I' I I.,/\ I I .\ O 3.. ,1, I mum—wremrw mfimsn ”Tm—35m O 0.2 0.4 0.6 0.8 Fig 119 (b) As discussed above, the 32P-labeling of phos- pholactate from 32P-OrthOphosphate depends upon 32P- orthOphOSphate incorporation into acetyl phosphate which is formed by enzymatic oxidation of lactate. Since acetyl phOSphate was shown to be the phOSphoryl donor in formation of acrylate (presumably the donor forms phos- pholactyl intermediate) (of. Figure 7) and since a more rapid labeling or the intermediate is desirable, 32P- acetyl phOSphate was synthesized in a manner identical to acetyl phosphate except about 0.1 mC 32P-orthophOSphate Iwas added (of. MATERIALS AND METHODS). Using 32P-acetyl phosphate, instead of ggmgg-32PnATP or 32P-orthophOSphate, the double-labeled compound could be demonstrated with shorter incubation times even though the Specific activ- ity of the prepared 32P-acetyl phosphate was low. Fur- thermore by following the time course of the labeling it was possible to show that the double-labeled compound appeared and disappeared during the course of an incuba- tion as is typical of an intermediate (Figure 11), i.e., (1) the intermediate accumulates faster than acrylate and (2) when most of the lactate has been converted to acrylate the levels Of the intermediate decreases. (2) Purification of the double-labeled intermediate. A further means of demonstrating that the double-labeled compound is an intermediate would be to reincubate it with extracts and to show its conversion to acrylate. The control in this case must preclude the possibility 120 .OH oHeme eH uenasoeeu me ueumHese eyes meHaeem ese .Nz Heuss comm pe mes sOHperosH .meeSHob Hepop H1 ow ebaw Op Hopes use ”AHe sea sHeposa we Hmv poenpxe mo H1 0 .epesamoss Hmpeoenmmm_m.:m.o ”doc Hmpeoe_me mm.o «ean eseHmSpee RN00 :3 ossuosesoeanmm so 01 00.0.0 :04. may .833 2.2.. m 30.0 “epepoeHme.m mH.o “wsH3OHHom esp mo uepmHmsoo enspHHe soHpoeen esa evenneosa Hmpeoe Immm use ceauepepoeH eosm ueHeneH ussoaeoo 0:» mo eeespseHeseha .HH ehstm 12']. ea.“ Wee @3523 mm on em 0N , n. 0. n o 000 ,II. 00am A. 000 . 0 0.0. . mem0< _ . A moIdnn memgwqm , 000.0... .. _ I 0000 mmOADIjmo mama ZO >Id_mm._.z_ _ meermmordoremo, w , .000 9.0 xmodfdmw Zo >Id_mm._.2_ . I MFEU< ow / / «9024 I ._ W cm .. meqeodn 00.. medrdmordl _ he 0T0 xmoIdmo< I. m._.<._.o<.._OIdmOIn_ omN_mMI._.Z>m >I_.A_m_Io 0.? . (SEI‘IOINII) BiV'lAHOV 139 Both substrates give a hydroxamate whereas only one product does. Thus there is a net disappearance in the kinase reaction. Many attempts at demonstrating such a lactate- dependent disappearance of acetyl phOSphate were unsuccess- ful. The variations attempted were with lactate and only catalytic amounts of acetyl CoA; with substrate amounts of ethyl lactate, lactyl pantetheine, and lactyl CoA. Extracts and the calcium phosphate eluate were examined for activity in all the variations. However the negative results may be SXplained by assuming that acetyl phOSphate ‘ is readily formed from lactate, e.g., assume for argument's sake: (1) acetyl phOSphate + lactyl CoA-————+ acetate + phOSpho- lactyl CoA. (2) phOSpholactyl CoA -———+ P1 + acrylyl CoA electron transport (3) acetate + P1 + acrylyl CoA + 2H- ; phOSphorylation propionyl CoA + acetyl phOSphate (4) i lactate-————0 é pyruvate + H- (5) % pyruvate + Q Pi—eé 002 + H° -I- g acetyl phOSphate (6) propionyl CoA + lactate-————+ lactyl CoA + propionate NET: i P1 + 1% lactate -———+ % acetyl phOSphate + 5 C02 + propionate. Indeed lactate usually slowed the loss of acetyl phOSphate as predicted by the above assumptions. Now if it is also 140 assumed that dinitrophenol uncouples the electron trans- port phOSphorylation of the third reaction, then the net reaction would become: 4‘: acetyl phosphate + 1% lactate—9 acetate + it P1 + % C02 + propionate. Now there should be a lactate-dependent disappearance of acetyl phOSphate! As shown in Figure 17 in the presence of 10'“ M_DNP, the predicted lactate dependence of acetyl phosphate was observed. The difference (between lactate present and absent) in the rate of acetyl phOSphate dis- appearance gives a Specific activity of 0.092 umole/min/ mg protein. F. Reversal of Dinitrophenol Inhibition by Acetyl Phosphate The initial studies of DNP inhibition of propionate formation showed reversal by ATP. Now that acetyl phos- phate is known to be the real phOSphoryl donor in forma- tion of the intermediate it must also reverse the effect of DNP. To dramatize the effect of the more direct donor, the experiment was performed with extracts which had been dialyzed 6 hr against 200 volumes of 0.05 M phOSphate buf- fer (pH 6.5) and 1 mM_DTT. The assays were done in the "acrylate" system. The results show that acetyl phOSphate is effective in reversing the DNP inhibition (Figure 18). This Observation thus adds further evidence that acetyl 141 .0895: 024. 32804: as uoflaoeeu me ossseHssosuE so? uopoees use mHebnepsH we ueboees one: meHseem .mz seuss comm we mes soapensosH .eeSHob Hepop H1 00m ewa on nope: use ”AHe Hen sHepOHa we mm «as m ueNhHeHuv poespwe no H1 on ”HosesaospHsHu.m #:0a ”400 Hmpeoe.me m.0 ”epepoeHme.m 3.0 n3.1.3.0020 issue a 30 ”0:3 0:30.203 $0.0 ”~30: as m 22.4. was wommsp mmmwm.a mm0.0 ”wsHsOHHom esp uesaeusoo enspHHe sOHpoeeh one HosesaoepHsHu_m IoH mo 00semena esp sH Onesaeosa Hmpeoe co moseneeasemHu psewseaeulepepoeH one .heeme eeest thoeq .mH enstm 0052.2 142 mm on _ um 0m 0. 0. 0 000.0 I m 9.0 G H O _ . X . 0009‘ 30 v 0.284.. W e V \ _ .. _ o _ . owd 3 O O O . o _ . + II . n_u. nWV O _ . _ _ I000 t e O a ( e _ . . . 000 mmFm0< 143 .mdthQCG oasQMHwOmeOHso mam How mamb lumpsa pm cmboamn mums mmaaamm .mz 900:: comm ad was nodpwnzonH .mfidd0b Hana» Ha oom.o mbdw op papa: dam “doc Hmpoom_ma a ”Aaa nmm camponn ma mm .Hg m vouhamauv pomnpxm mo H1 om ”Honosnonpacau flwdloa “nosed ahnosmmosa a 90.0 ”3337mm m mmoé 3:3 3323a “No.0 “mam: ma N. 2mm.“ m3 nmmmsn mummm.m mmo.o ”wadsoaaom on» omcdmpcoo whapuaa manna msa scapdnanna Hozmnmonpdcav no mehmbmh mpmnnmonn thmod .wa onuwam U. u. 1.. mw._.32=>_ mm on mm ON 2 _ 9 0 . flo _ _ V . a _/ ._... mzoz w .n_._.< _ . . .l. o . . V n_v _ . ow i . _ . . 3 \ . 1 . om .W . , . . 0 0m. Maw v OO._ ZO_._._m_IZ_ JOZMIQOthE “.0 44mmm>wm 11+5 phOSphate is the phosphoryl donor in formation of an intermediate (presumably algha-phOSpho-Q-lactyl CoA). CHAPTER V W The central finding of this research was the fact that phospholactyl CoA is an intermediate between lactyl CoA and acrylyl CoA in the pathway of propionate forma- tion from lactate. This finding is a consequence of the following observations: (1) 18O is transferred from 2-180-lactate to orthophosphate concomitant with prOpion- ate formation; (2) a double-labeled intermediate accumu- lates during incubation of lac-lactate and 32P-aoetyl phosphate; (3) the labeled intermediate is converted to acrylate; and (h) the labeled intermediate was confirmed to be phospholactate by alkaline phoSphatase treatment which released equimolar amounts of lactate and phoSphate. Whereas a phospholactyl CoA intermediate contra- dicts Baldwin's simple dehydration reaction catalyzed by lactyl CoA dehydrase, it does eXplain his failure to observe the interconversion of lactyl CoA and acrylyl CoA by a direct spectrophotometric assay. It also eXplains the sudden loss of activity upon purification (Baldwin, 1962). Furthermore a phospholactyl CoA intermediate dovetails very well with Ladd and Walker's observation of the ability of dinitrophenol to inhibit the lactate- acrylate interconversion and its reversal by ATP or 1u7 acetyl phosphate (Ladd and Walker, 1965). The elimination of the phosphate from phospholactyl CoA to form acrylyl CoA presumably by a phoSpholactyl CoA lyase represents another example of phosphate-facilitated leaving of a hydroxyl group. The other cases of enzymatic reaction of similar reaction mechanism are (1) threonine synthetase and (2) ATP:5-pyrophoSphomevalonate carboxy- lyase: (ing-o ® 0 ® ([332-0 ® 0 ® c'mz-o ® 0 ® CH CH CH I 2 ATP I 2 -C02 I 2 HO-C-CHB : (9-0-0-033 4 0.033 I I -P 1 I .. l - coo coo MEVALONIC ACID ISOPENTENYL PYBOPHOSPHATE PYROPHOSPHATE 000' 000‘ I 18 l HZN-cn H2 0 ‘ HZN-clza8 fife-H «1D 1 ' Hcl:1 o-a CHZ-O P 033 g-PHOSPHO THREON INE HOMOSERINE Model system studies have also shown that the phosphoryl group enhances elimination reactions (Cherbuliez 22 3;.. 1962). Uniquely acetyl phosphate is the phosphoryl donor in the case of g, elsdenii. With few exceptions, enzyme- 148 catalyzed phosphorylations involve nucleoside triphos- phates as the phosphorylating agents. Acetyl phoSphate has been found to be the donor in formation of Qgglucose- 6-phosphate from Q-glucose as catalyzed by an enzyme from Aerobacter aerggenes (Kamel and.Anderson, i96h). In this case the enzyme also utilizes hexose phosphates as donors, e.g., Qrmannose-é-phosphate, and other compounds such as carbamyl phosPhate and phoSphoramidate (Kamel and.Anderson. 1967), leading to the conclusion that this is a nonspecific phosphotransferase which can utilize acetyl phoSphate as well as other donors. Studies with Clostridium kluzgeri which is an anaerobe like 2, elsdenii have demonstrated the phosphoryl-donating prOperties of acetyl phOSphate in several reactions (Decker, 1959). Thus the use of acetyl phOSphate to form phospholactyl CoA is not an unprecendented example of its use as a phoSphoryl donor. A very important aspect of this new reaction sequence is the implication of the present results with respect to the possibility of electron transport mediated phoSphory- lation in the anaerobe g, elsdenii. Inasmuch as an acetyl phosphate, a potential source of ATP. is consumed during formation of phoSpholactyl CoA, an additional phoSphoryla- tion besides that of the phoSphoroclastic system must occur otherwise the organism would not be able to grow: (with electron transport mediated phosphorylation) i. lactate + X CoA—9 lactyl CoA + X 2. 3. 1+. 5. 6. 7. 8. 9. 10. NET: 1&9 lactyl CoA + acetyl P——a phospholactyl CoA + acetate phospholactyl CoA ——)P1 + acrylyl CoA Y + P + 2 H + acrylyl CoA -—» Y~P + propionyl CoA i Y~P + acetate ——-> acetyl P + Y lactate —-> pyruvate + 2 H pyruvate + CoA ——) H21 + 0021‘ + acetyl CoA acetyl CoA + P1 -——9 acetyl P + CoA ADP + acetyl P ———> acetate + ATP propionyl CoA + X ——> propionate + X CoA 2 lactate + ADP—4; prOpionate + acetate 4» C02 + (without electron transport mediated phosphorylation) 1. lactate + X CoA———> lactyl CoA + X lactyl CoA + acetyl P —-> phospholactyl CoA + acetate phospholactyl CoA—9 P1 + acrylyl CoA 2 H + acrylyl CoA——> prOpionyl CoA lactate —-—>pyruvate + 2 H pyruvate + CoA—+321 + 0021 + acetyl CoA acetyl CoA + Pi—> acetyl P + CoA NET: 2 lactate —> prOpionate + acetate + C02 + H2 +- . The existence of anaerobic electron transport phos- phorylation has been speculated for some time. There is precedent for this sort of phosphorylation (E. R. Stadtman, 1966). Direct evidence of anaerobic ATP generation in 150 clostridia by a mechanism which does not involve substrate phosphorylation was found in the reductive deamination of glycine as in Clostridium sticklandii and Clostridium lentOputrescens (Stadtman and Elliott. 1956). The system was resolved into an electron tranSport protein, ferredoxin. an acidic and low molecular weight protein, and a quinone (Stadtman 22H§;.. 1958: Stadtman. 1962: Stadtman. 1966): NADH + H& + glycine + P + ADP-——-9>NAD+ + NH3 + ATP + acetate 1 In the similar case of Clostridium aminobutyricum growth studies have shown that 7.6 mg of dry cells are derived from 1 mmole of gaggg—aminobutyrate compared to 5.0 mg which would be expected from the substrate-level phosphor- ylation reactions predicted to occur. Second, in the case of anaerobic streptococci, electron transport phOSphorylation has been implicated. Streptococci are facultative anaerobes which do not possess cytochromes. Thus any electron transport mediated phoSphorylation must be different from that which occurs in mitochondria. Studies with Streptococcus faecalis have revealed growth beyond the limits of the substrate-level phosphorylation reactions known to occur. and the addi- tional growth suggests a P/O ratio of 0.6 (Smalley g§_§;.. 1968). In‘§. agalactiae, ATP formation has been demon- strated with cell-free extracts: P + NADH + ADP + 02 ————4»ATP + 320 + NAD. i 151 With respect to the equation above it should be pointed out that the usual acceptor is nitrate not oxygen and that oxygen was used to facilitate assay (oxygen uptake was determined manometrically). In this system a P/O ratio of 0.15-0.#2 was observed (Mickelson, 1968). Preliminary studies with.§, elsdenii suggest the existence of a soluble, electron transfer system derived from the coupling of the lactate dehydrogenase with the acyl CoA dehydrogeanse: lactate acrylyl CoA I\ egg—\l pyruvate prOpionyl CoA The enzymes are not precipitated by ultracentrifugation for several hours at greater than 100,000 times gravity. Further evidence that the system is not particulate is that solubilizing agents, such as glycerol, and phospho- lipids are uniformly inhibitory (cf. APPENDIX, evidence for soluble system). Whether phOSphorylation accompanies the electron transfer described above is not known. However electron transfer from the lactate dehydrogenase to the acyl CoA dehydrogenase cannot be tightly coupled inasmuch as the fermentation balance reveals that for growth on 100 mmole of lactate 71 mmole of acetate is formed (though most of it is converted to higher fatty acids, especially butyrate and valerate) and 39 mmole of propionate is formed (half of it is converted to 152 valerate). Inotherwords, the pathway to acetate operates twice for every time that to propionate does (Elsden 32 al., 1956). Determination of ATP formation by means of the usual hexokinase trap (Pinchot, 1957) as a method of verifying electron transfer phosphorylation is precluded by the presence of a very active adenylate kinase (Baldwin and Milligan, 1964). An alternate approach would be to: (1) isolate the components of the electron transport system: (2) reconstitute the system: and (3) isolate. coupling factor which possibly would restore phosphoryla- tion. Finally, Judging from the available data, the basic metabolic system for lactate utilization in Z, elsdenii is as shown in Figure 19: thus the net reaction for extracts is: 2 lactate + ADP + P1-————a>propionate + acetate + 0021 + HZT + ATP. 3 U “ascends .m 5 203.3335 opmpoma no.“ acpmmm oaaopmpoa 3me 23. .m« ondwfim q «twee. assse_ Essential F m: Emu; e04 nIo nxo i nzo M . ......o A u @o- ..o. A A N zoo: . _ zoow I 0" + SE zooo :08 E ... 223° n. .236 at» E .+> my > W .UDN av a use . . use $.33 . E §§dotd gazedotd use 89E? e ..otdmcrd «so .233 333. e...o ~:o e_._o eIo n w . = . m Io Io , Io IA._V0@ \thooo 100: :0 1.0 A AWL A _ _ «com- ..o. «com- ..o Ami «com- ..o A 48?. o Ioo: . e 388 _. _ :08 o m o o :08 ACD AchAD ATP EAL CoA CoA-T DTT Glc INT LDH MB NAD(H) NADP(H) PEP P1 PMS PMSF rds THF TPP ABBREVIATIONS USED acyl CoA dehydrogenase‘ 3-acetylpyridine adenine dinucleotide (analog of NAD) alcohol dehydrogenase adenosine triphosphate British anti-Lewisite or 2,3-dimercaptopropanol coenzyme A coenzyme A transferase Cleland's reagent or dithiothreitol glucose 2- ara-iodophenyl-3-para-nitrophenyl-S-phenyl- te razolium chloride lactic acid dehydrogenase methylene blue (reduced) nicotinamide adenine dinucleotide (reduced) nicotinamide adenine dinucleotide phOSphate phOSphoenolpyruvic acid orthophosphate phenazine methosulfate “ FU 306.34 phenylmethylsulfonylfluoride, a proteinase inhibitor similar to diisopropylfluorophosphate but not poisonous rate determining step tetrahydrofuran, a water miscible ether thiamine perphoSphate (vitamin B1) 155 APPENDIX CALCULATION OF MINIMUM SPECIFIC ACTIVITY OF THE ENZYMES OF THE ACRYLATE PATHWAY During any one time interval: (E) (At) = X where E is enzyme activity in umole/min/mg protein, t is time in minutes, and X is the amount of material passing through the pathway in umole/mg protein. Summing the equation over the entire fermentation period gives an integral 720 05 E(t) dt = X Before the integral can be evaluated E as-a function of time, E(t), and X must be known. Determination of E(t) Since the number of cells double each generation and if it is assumed that the amounts of the enzymes of the pathway double in a similar manner, then: E e 30(2)n t where Et is the enzyme activity at any time t and n is the number of generations. Now, if a 5% inoculum is used, the initial amount of enzyme is: E0 = 0.05 Ef 156 157 and number of generations can be calculated: E e E e 0.05 Ef (2)n final f 1.00 = 0.05 (2)n .1. .05 = 20 = 2n log2 (20) = n n = “.31 The division time is 720/4.31 or 167 min. Now E can be expressed as a function of time: LP. 31 t E (t) = 0.05 Ef (2) Remember Ef is final enzyme activity and is not a variable. Determination of X Twenty liters of medium containing 270 g of lactic acid when fermented by 2, elsdenii yield, among other products, 0.36 moles of propionate and 0.46 moles of val- erate (Gutierrez gtflgl., 1956). Both propionate and val- erate are products of one pass through the direct reduc- tive pathway: hence, during the whole fermentation at least 0.81 moles (0.36 + 0.46 = 0.82) must represent direct reductive pathway activity. With a five percent inoculum the bacteria reach stationary phase in about twelve hours. Thus the pathway must be as active as 820 000 moles 2 A- 41 umole/ml per 1 hr 20.000 ml _ 41. 158 A deep culture of twenty liters of P, elsdenii produce 5,250 mg of protein (average of 25 determinations) or 0.2625 mg protein/ml. Thus the minimum activity is X = 41 mole ml 156 umole/mg protein per 12 hr FLA—0.2625 mg/ml = 156). Integration Substituting into the integral gives 72o 72%—L" 1 t g 0.05 Ef (2) dt = 156 O 01‘ 720 S (2)0°00599 1‘ dt = .416— 0.05 Ef which is in the form au au g du = _2____-+ c b a 1n b Hence . 2 . 156 = (2)0 00599(7 o) _ (2)0 00599(o) 0.05 Ef .00599 1n 2 .00599 In 2 = 12.2 _ 1.00 .00414 .00414 = 4800 - 242 = 4558 or 116 = Br (. 05) (4558) Ef = 0.684 umole/min/mg protein. 159 CALCULATION OF EXPECTED BHLlLl'CfiRATIO The tritium used gave 1.16(10)10 counts per 0.020 ml: in incubation system 1 (cf. MATERIALS AND METHODS) the hydrogens of the water then had a Specific activity of: 1.16i19110 £22.1l§.5£22l21 = 8.03(10)10 cpm/mole 2.6 g The lactate had a Specific activity of: 63959 cpm = 5,33(1o)8 cpm/mole 0.12 x 10'3 moles Hence the expected ratio in propionate was: 10 W = 301 5.33(10)8 . STABILIZATION 0F EXTRACTS BY PROTEINASE INHIBITOR Activity of extracts in producing propionate from lactate deteriorated upon storage, especially when dival- ent metals such as Ca2+ were present. Such behavior has the appearance of proteolytic digestion. A proteinase inhibitor phenylmethylsulfonylfluoride (PMSF) was effec- tive in preventing this deterioration (APPENDIX Figure 1). It was added to extracts at time of preparation according to the procedure of Steinman and Jakoby (1967). PMSF is better than diisopropylfluorophosphate because it is not detrimental to the nervous system yet has potency in inhibiting proteinases. 160 .mpmo esp mo psoapmcap meamsvw pmdoa m psomoaaeh cacao moEHH one .maomamz QZE.mA4HmmB