A catalytic role for histidine 237 in rat mammary gland thioesterase II.

Abstract

The involvement of a histidyl residue in the catalytic mechanism of thioesterase II, a serine active-site enzyme that catalyzes the chain terminating reaction in de novo fatty acid synthesis, has been inferred from studies with the inhibitor diethyl pyrocarbonate. Its likely location has been predicted by identification of conserved residues in related thioesterases and ultimately confirmed by site-directed mutagenesis. Diethyl pyrocarbonate inactivated the enzyme with a second-order rate constant of 49 M-1 s-1 at pH 6, 10 degrees C. Data analysis indicated that although several residues reacted with the reagent, modification of a single residue was responsible for the inactivation. Removal of a single ethoxycarbonyl moiety by treatment with neutral hydroxylamine completely restored enzyme activity. Prior ethoxycarbonylation of the histidyl residue blocked the ability of the active-site serine to react with phenylmethanesulfonyl fluoride. Comparison of the amino acid sequences of five structurally related proteins indicated that only 1 histidine has been completely conserved. Replacement of this residue in rat thioesterase II (His-237) with arginine and leucine by mutagenesis reduced the catalytic activity by 2-3 orders of magnitude. The activity of the mutant thioesterases, unlike that of the wild-type enzyme, was relatively insensitive to inhibition by diethyl pyrocarbonate and phenylmethylsulfonyl fluoride. These studies provide strong evidence that His-237 is involved directly in catalysis and suggest that its role is to increase the nucleophilic character of the active-site Ser-101 by acting as a proton acceptor thus facilitating acylation of the seryl residue. The mechanism appears to share certain common features with the charge-relay system characteristic of other esterases.