Abstract:
:Although the short-chain dehydrogenase/reductase (SDR) superfamily contains a very large number of members defined in annotated databases and by biochemical and structural studies, very few SDR enzymes have been identified that have a homologous partner catalyzing the same reaction but with an opposite stereospecificity. In the present study we have cloned and expressed one of these enzymes, the 2-[(R)-2-hydroxypropylthio]ethanesulfonate (R-HPC) dehydrogenase, that is part of the coenzyme M-dependent pathway of alkene and epoxide metabolism in Xanthobacter strain Py2. Investigation of the kinetic mechanism using product inhibition suggested that a compulsory-ordered ternary complex mechanism was followed. The pH dependence of k(cat)/K(m) indicated the presence of a single ionizable residue of catalytic importance (pK(a) = 6.9) that was proposed to be Y155 of the catalytic triad. Amino acid substitutions of the putative catalytic triad residues produced inactive enzymes (S142C, Y155F, Y155E, and K159A) or enzyme with a greatly decreased activity (S142A). Inhibitors were investigated as probes of the molecular features of R-HPC that contribute to substrate binding. 2-[(S)-2-Hydroxypropylthio]ethanesulfonate (S-HPC) and 2-(2-methyl-2-hydroxypropylthio)ethanesulfonate were found to be competitive inhibitors of R-HPC with K(ic) values close to the K(m) for R-HPC. The arginine-specific modifiers 2,3-butanedione and phenylglyoxal were found to be inactivators, and inactivation could be protected against by the addition of R-HPC. 2,3-Butanedione was found to reduce enzyme activity with R-HPC as a substrate much more dramatically than with substrates that lacked a sulfonate moiety [e.g., 2-propanol, (R)-2-pentanol, and (R)-2-heptanol]. Amino acid analyses of enzyme modified by 2,3-butanedione in the presence and absence of S-HPC suggested protection of a single arginine residue. On the basis of these results, we propose that one or more active site arginines play a key role in substrate binding via an ionic interaction with the sulfonate moiety of R-HPC.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Clark DD,Ensign SAdoi
10.1021/bi0118005subject
Has Abstractpub_date
2002-02-26 00:00:00pages
2727-40issue
8eissn
0006-2960issn
1520-4995pii
bi0118005journal_volume
41pub_type
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