Uber das Katalyseprinzip der Malat-synthase (1967)

Malate-synthase catalyzes an aldol condensation: The enzyme enolizes acetyl-CoA and hydrolyzes malyl-CoA. I. Acetyl-CoA-enolase, “monofunctional”. Enolization of acetyl-CoA by malate-synthase was demonstrated by isotopic-exchange between the methyl hydrogen of the acetyl group and tritiated water. [3H]Acetyl-CoA was determined as [3H] p-nitroacetanilide. Under optimal conditions, the rate of enolization was approximately a thousand times slower than that of the synthesis of malate. The isotopic exchange was dependent on high Mg++ and enzyme concentrations, pH and on long incubation times. Mg++ ions could be partially replaced by other divalent metal ions. The action of the metal ions is considered an acid catalysis, i.e. the thioester carbonyl when bound to the Lewis acid becomes polarized, the methyl hydrogen becomes acidic. The fact, that the related enzyme citrate-synthase required base-catalysis for the enolization of acetyl-CoA, suggested that a partial mechanism was demonstrated with each enzyme (acid- and base-catalysis) both of which are active cooperatively in both the enzymes. The chemical model of this cooperative action is the bifunctional catalyst of Swain and Brown. With malate-synthase it was therefore attempted to demonstrate the participation of the carboxylate anion of glyoxylate as a base-catalyst in the enolization of acetyl-CoA, using substrate-analogues of glyoxylate. II. Acetyl-CoA-enolase, “bifunctional”. α-Ketoacids stimulated the rate of enolization in the order pyruvate > oxalacetate ≫α-ketobutyrate > α-ketoglutarate ≅α-ketovalerate, i.e. the more, the greater the structural similarity to glyoxylate. Other carboxylic acids were inactive. Tritio-acetyl-CoA-yielding side reactions were excluded. Pyruvate inhibited the synthesis of malate competitively (Ki= 10−3 M). The affinity of pyruvate for the enzyme yielded ordinary kinetics (Km= 10−3 M) as determined by the isotopic-exchange. No sign of aggregation or dissociation of the enzyme was detectable, as judged from sedimentation studies with and without acetyl-CoA and pyruvate. Taken together these results exclude an allosteric action of the α-ketoacids and agree with their action as base-catalysts. The fact, that citramalyl-CoA, the condensation product of acetyl-CoA and pyruvate, was not attacked by the enzyme, suggested that only the carboxylate anion of the α-ketoacids participated in the enolization. Due to steric hindrance at the active site, the ketocarbonyl of these acids is in a position too far removed for reaction with the acetyl-CoA carbanion formed. The α-ketoacids therefore induce the isotopic exchange. The rate of enolization in the presence of pyruvate was stimulated a thousand times and was nearly equal to that of the synthesis of malate. As in the chemical model both the nucleophilic and the electrophilic groups act cooperatively: removal of either Mg++ or pyruvate abolished the enolization. Taken all together, these results provide proof for the participation of the carboxylate anion of glyoxylate in the enolization of acetyl-CoA in the natural system. III. Malyl-CoA-hydrolase. A coupled optical test was used for the enzymatic hydrolysis of malyl-CoA, in which the hydrolysis of the substrate with the subsequent oxidation of CoA-SH by ferricyanide was determined from the decrease of absorption. Both the affinity and turnover number of malyl-CoA when used as a substrate, were approximately 10−3 times less than those of the natural reactants. This is in agreement with the existence of enzyme bound malyl-CoA in the natural system. The enzyme required Mg++ and catalyzed the hydrolysis of (S)-malyl-CoA faster than that of the diastereomeric mixture and of the (R)-diastereomer. IV. Rate Determining Step. Malate, when synthesized in tritiated water in the absence of pyruvate contained no tritium; acetyl-CoA after partial reaction with glyoxylate remained unlabelled: the enolization of acetyl-CoA is the rate-determining step in the synthesis of malate. V. Equivalence of the Methyl Hydrogens in Enolization and Synthesis. In the equilibium of the isotopic exchange, the specific (sA) acitivity of p-nitroacetanilide, corrected for losses during its isolation, corresponded to sA (acetyl-CoA) = 0.94 × sA(H2O). Theoretically it should be sA (acetyl-CoA) = 3/2 sA(H2O) without mass effect, since the methyl hydrogens are chemically equivalent. The value obtained experimentally may reflect a thermodynamic isotopic effect and may also indicate stereospecific exchange of two methyl hydrogens with tritium. The latter was excluded by using chemically prepared [3H-2C]acetyl-CoA, which, in the presence of pyruvate and malate- synthase in water lost its tritium content completely. [3H]malate, synthesized enzymatically from either chemically or enzymatically prepared tritio-acetyl-CoA was recognized as (S)-2- hydroxy-3,3-ditritio-succinate by the isotopic exchange catalyzed by fumarase. As expected, the methyl hydrogens of the acetyl group are thus equivalent in the reaction with either pyruvate for the enolization, or glyoxylate for the enolization and subsequent synthesis of malate. VI. Principle of Catalysis. The following results illustrate that the principle of catalysis is the approximation of the reactants: a) No acetyl-enzyme is formed between acetyl-CoA and malate-synthase. b) Cooperative catalysis with Mgff and the carboxylate-anion of glyoxylate generates enolic acetyl-CoA. c) In the natural system this immediately reacts with the aldehyde-carbonyl of glyoxylate to form (S)-malyl-CoA. d) The enzyme catalyzes the hydrolysis of (S)-malyl-CoA used as a substrate. e) The chemical hydrolysis of substituted succinyl-monothioesters in neutral aqueous medium is facilitated by neighboring group participation and proceeds via substituted succinic-anhydride. f) The bifunctional enzymatic catalysis corresponds in every respect with the bifunctional chemical catalysis. Proper orientation of the acidic and basic groups in the chemical model can be achieved by their insertion into one molecule, and in the enzymatic catalysis by their corresponding orientation on the enzyme. The facts, that Mg++ was required for the enolization of acetyl-CoA as well as for the hydrolysis of malyl-CoA, and that the enolization was abolished when either acid- or base-catalyst was removed, indicate the formation of a complex between Mg++ and the substrates on the enzyme. In this complex the reactants with both their carbonyls are coordinatively bound to the Lewis acid and then by a forced intramolecular reaction are converted to the products. VII. Related Enzyme Catalyses. In the related catalyses the Mg++ may be replaced by a Lewis acid constituitively present in the protein, and the ketoacid may be arranged sterically opposite to the position of either glyoxylate on malate-synthase or oxalacetate on citrate-synthase. The carbonyl group is then attacked from the R-side by the acetyl-CoA-carbanion and the (R)-hydroxyacid is formed as the product. acetyl-CoA.