STUDIES ON (-)-CITRAMALIC ACID FORMATION BY RESPIRATION-DEFICIENT YEAST MUTANTS

V. PURIFICATION AND SOME PROPERTIES OF CITRAMALATE CONDENSING ENZYME

Abstract

Citramalate condensing enzyme has been purified 95-fold by ammonium sulfate fractionation, and by Sepharose 4B and DEAE-cellulose column chromatography from RD petite mutant strain of Saccharomyces carlsbergensis. On the basis of the fact that (-)-citramalate fraction had all of the radioactivity while (+)-citramalate fraction contained no radioactivity when enzymically formed radioactive citramalic acid with authentic carrier DL-citramalic acid was subjected to optical resolution, it was made clear that this enzyme catalyzed the formation of (-)-citramalate from pyruvate and acetyl-CoA. The optimal pH of the enzyme was 7.4 and the K_m value for pyruvate was 2.3×10^-3M. The purified enzyme preparation still has an activity toward α-ketobutyrate and α-ketoisovalerate. The K_m value for α-ketoisovalerate was 2.6×10^-5M. L-Leucine inhibits to the same extent the respective condensation reactions between these three α-keto acids and acetyl-CoA. α-Ketoisovalerate is an effective inhibitor of citramalate condensing reaction. The enzyme was strongly inhibited by p-chloromercuribenzoate, Cu²⁺, Zn²⁺, Hg²⁺, Pb²⁺, and Cd²⁺. Citramalate condensing enzyme appears to be identical with α-isopropylmalate synthetase in the leucine biosynthetic pathway. The role of this enzyme in RD petite mutant strains of Saccharomyces carlsbergensis was discussed in relation to citramalate formation.