Isolation and Function of the Subunits of Citramalate Lyase and Formation of Hybrids with the Subunits of Citrate Lyase

Abstract

1. The enzyme complex citramalate lyase from Clostridium tetanomorphum, composed of six copies of three different proteins, has been dissociated into its protein components and these were isolated in an enzymically active state. Citramalate lyase has been reconstituted from the isolated subunits. 2. The enzyme complex consists of an acyl carrier protein and two different enzymes, the α and β subunits. The isolated enzymes were active if acetyl‐thio‐acyl carrier protein and (3S)‐citramalyl‐thio‐acyl carrier protein were substituted for the corresponding acyl‐CoA derivatives. 3. The α subunit in the presence of (3S)‐citramalyl‐CoA and acetate catalyzed the formation of acetyl‐CoA and citramalate. The β subunit catalyzed the cleavage of (3S)‐citramalyl‐CoA to acetyl‐CoA and pyruvate. This reaction was dependent on Mg2+ and was abolished if EDTA was added in excess. It was concluded from these results that the α subunit represents an acetyl‐thio‐acyl carrier protein: citramalate acyl carrier protein transferase within the native complex, and the β subunit a (3S)‐citramalyl‐thio‐acyl carrier protein lyase. 4. Results similar to those given above have previously been obtained with the enzyme complex citrate lyase from Klebsiella aerogenes, but with citrate, (3S)‐citryl‐thio‐acyl carrier protein or (3S)‐citryl‐CoA instead of citramalate and its corresponding derivatives. Thus, not only molecular weight, subunit composition and chemical events during substrate turnover are nearly identical in the two different enzyme complexes but also the functions of the individual subunits. 5. The close relationship between the two different enzymes from the two different microorganisms was confirmed by cross reactions between the isolated subunits of each enzyme complex producing a hybrid enzyme complex. Copyright © 1977, Wiley Blackwell. All rights reserved