p-Chloromercuribenzoate specifically modifies thiols associated with the active sites of ?-Ketoadipate enol-lactone hydrolase and succinyl CoA: ?-Ketoadipate CoA transferase

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Abstract
β-Ketoadipate enol-lactone hydrolase (EC 3.1.1.24) and succinyl CoA:β-ketoadipate transferase (EC 2.8.3.6) catalyze consecutive metabolic reactions in bacteria. The enzymes appear to be members of different families of related proteins. Enzymes within the enol-lactone hydrolase family appear to have diverged so extensively that common ancestry sometimes is not directly evident from comparison of NH2-terminal amino acid sequences of the proteins. Amino acid sequences at or near the active sites of the enzymes are likely to have been conserved, and hence a chemical proble that reacted specifically near the active sites of the enzymes might identify regions of amino acid sequence in which evolutionary affinities among widely divergent proteins could be identified. p-Chloromercuribenzoate appears to be such a probe because enol-lactone hydrolases and CoA transferases from Acinetobacter calcoaceticus and Pseudomonas putida were completely inhibited by stoichiometric quantities of the compound which appears to modify selectively cysteinyl side chains at or near the active sites of the enzymes. Stoichiometric inhibition of P. putida enol-lactone hydrolase was observed in the presence of excess dithiothreitol; therefore the reactive cysteinyl residue in this enzyme appears to be nucleophilic. The hydrolase is inhibited by β-ketoadipate, but the compound must be supplied at 10 mM concentrations in order to achieve 50% inhibition, so the product inhibition is unlikely to be significant under physiological conditions.