Abstract
An enzyme that catalyzes the synthesis of S-carboxymethyl-L-cysteine from 3-chloro-L-alanine (3-Cl-Ala) and thioglycolic acid was found in Escherichia coli W3110 and was designated as S-carboxymethyl-L-cysteine synthase. It was purified from the cell-free extract to electrophoretic homogeneity and was crystallized. The enzyme has a molecular weight of 84, 000 and gave one band corresponding to a molecular weight of 37, 000 on SDS-polyacrylamide gel electrophoresis. The purified enzyme catalyzed the β-replacement reactions between 3-CI-Ala and various thiol compounds. The apparent Km values for 3-Cl-Ala and thioglycolic acid were 40 mM and 15.4mM. The enzyme showed very low activity as to the α, β-elimination reaction with 3-Cl-Ala and L-serine. It was not inactivated on the incubation with 3-Cl-Ala. The absorption spectrum of the enzyme shows a maximum at 412 nm, indicating that it contains pyridoxal phosphate as a cofactor. The N-terminal amino acid sequence was determined and the corresponding sequence was detected in the protein sequence data bank, but no homogeneous sequence was found.
