The Enzyme Complex Citramalate Lyase from Clostridium tetanomorphum

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Abstract
1 The enzyme citramalate lyase from Clostridium tetanomorphum is not stable in crude extracts. However, the inactive enzyme can be reactivated by incubation with dithioerythritol followed by acetylation with acetic anhydride. Reactivation was also obtained with acetate, ATP. MgCl2 and acetate :SH‐enzyme ligases (AMP) from C. tetanomorphum or Klebsiella aerogenes. 2 Incubation of the inactive enzyme with iodoacetate resulted in rapid loss of enzymic activity as determined by reactivation with acetic anhydride whereas the active enzyme was stable in the presence of iodoacetate. Using iodo[2‐l4C]acetate the sites of carboxymethylation and acetylation where identified as cysteamine residues of the enzyme. The results demonstrate that the active enzyme contains acetyl thiolester residues which play the central role in the catalytic mechanism. 3 Citramalate lyase was purified by a procedure almost identical to that already described for citrate lyase from K. aerogenes. The molecular weight of citramalate lyase is equal to that of citrate lyase (Mr= 5.2–5.8 × 105) as estimated by gel chromatography and sucrose gradient centrifugation. Polyacrylamide gel electrophoresis of citramalate lyase in sodium dodecylsulfate yielded three polypeptide chains (Mr: α 5.3–5.6 × 104; β 3.3–3.6 × 104; γ 1.0–1.2 × 104) in probably equal molar amounts. These data lead to a hexameric structure (α,β,γ)6 of the complete enzyme. 4 Pantothenate (5 mol/mol of enzyme) and the essential cysteamine residues were exclusively present in the γ‐chain, the acyl carrier protein of citramalate lyase. The acyl exchange and cleavage functions, probably catalysed by the α and β‐subunits, were measured with acyl‐CoA derivatives which were able to substitute for the natural acyl carrier. 5 The results demonstrate that citramalate lyase is an enzyme complex with structure and functions closely resembling those of citrate lyase. Although the similarity between citramalate lyase and citrate lyases from various organisms suggests a close evolutionary relationship, these occur in very different, unrelated bacteria. A parallel situation found in the distribution of the nitrogenase system among procaryotes is discussed