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).
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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Evidence ID | Analyze ID | File | Description |
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