Malate synthase: proof of a stepwise Claisen condensation using the double-isotope fractionation test

Journal Article · Tue Aug 09 04:00:00 EDT 1988 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00416a020· OSTI ID:7188328

Clark, J D; O'Keefe, S J; Knowles, J R

Although aldolase-catalyzed condensations proceed by stepwise mechanisms via the intermediacy of nucleophilic enol(ate)s or enamines, the mechanisms of those enzymes that catalyze Claisen-type condensations are unclear. The reaction pathway followed by an enzyme from this second group, malate synthase, has been studied by the double-isotope fractionation method to determine whether the reaction is stepwise or concerted. In agreement with earlier work, a deuterium kinetic isotope effect /sup D/(V/K) of 1.3 +/- 0.1 has been found when (/sup 2/H/sub 3/)acetyl-CoA is the substrate. The /sup 13/C isotope effect at the aldehydic carbon of glyoxylate has also been measured. For this determination, the malate product was quantitatively transformed into a new sample of malate having the carbon of interest at C-4. This material was decarboxylated by malic enzyme to produce the appropriate CO/sub 2/ for isotope ratio mass spectrometric analysis. The /sup 13/C isotope effect with (/sup 1/H/sub 3/)acetyl-CoA is 1.0037 +/- 0.0004. By use of the known values of the intermolecular and intramolecular deuterium effects and of /sup 13/(V/K)/sub H/, the value of the /sup 13/C isotope effect when deuteriated (/sup 2/H/sub 3/)acetyl-CoA is the substrate can be predicted for three possible mechanisms. The results show clearly that the two salient characteristics of enzymes that catalyze Claisen-like condensations, namely, the absence of enzyme-catalyzed proton exchange with solvent and the inversion of the configuration at the nucleophilic center, which had been suggestive of a concerted pathway, are not mechanistically diagnostic.

• Research Organization: Harvard Univ., Cambridge, MA (USA)
• OSTI ID: 7188328
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:16; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL PATHWAYS
CARBON 13
CARBON ISOTOPES
COENZYMES
DEUTERIUM COMPOUNDS
ENZYMES
EVEN-ODD NUCLEI
HYDROGEN COMPOUNDS
HYDROGEN TRANSFER
ISOTOPE EFFECTS
ISOTOPES
KINETICS
LIGHT NUCLEI
LYASES
NUCLEI
REACTION KINETICS
STABLE ISOTOPES