A role for His-160 in peroxide inhibition of S. cerevisiae S-formylglutathione hydrolase: evidence for an oxidation sensitive motif.

While the general catalytic mechanism of the widespread serine hydrolase superfamily has been documented extensively, much less is known about its varied modes of functional modulation within biological systems. Under oxidizing conditions, inhibition of Saccharomyces cerevisiae S-formylglutathione hydrolase (SFGH, homologous to human esterase D) activity is attributable to a cysteine (Cys-60) adjacent to its catalytic triad and approximately 8.0 Å away from the Oγ of the nucleophilic serine. Cys-60 is oxidized to a sulfenic acid in the structure of the Paraoxon-inhibited W197I variant (PDB 3C6B). The structural snap-shot captured an unstable reversibly oxidized state, but it remained unclear as to whether the oxidation occurred before, during, or after the reaction with the organophosphate inhibitor. To determine if the oxidation of Cys-60 was functionally linked to ester hydrolysis, we used kinetic analysis and site-directed mutagenesis in combination with X-ray crystallography. The essential nature of Cys-60 for oxidation is demonstrated by the C60S variant, which is not inhibited by peroxide in the presence or absence of substrate. In the presence of substrate, the rate of inhibition of the WT SFGH by peroxide increases 14-fold, suggesting uncompetitive behavior linking oxidation to ester hydrolysis. Here we found one variant, H160I, which is activated by peroxide. This variant is activated at comparable rates in the presence or absence of substrate, indicating that the conserved His-160 is involved in the inhibitory mechanism linking ester hydrolysis to the oxidation of Cys-60. Copper chloride inhibition experiments show that at least two metal ions bind and inhibit both WT and H160I. A structure of the Paraoxon-inhibited W197I variant soaked with CuCl(2) shows density for one metal ion per monomer at the N-terminus, and density around the Cys-60 sulfur consistent with a sulfinic acid, Cys-SO(2). A Dali structural similarity search uncovered two other enzymes (Bacillus subtilis RsbQ, 1WOM and Clostridium acetobutylicum Lipase-esterase, 3E0X) that contain a similar Cys adjacent to a catalytic triad. We speculate that the regulatory motif uncovered is conserved in some D-type esterases and discuss its structural similarities in the active site of human protective protein (HPP; also known as Cathepsin A).
(Published by Elsevier Inc.)