Identification of the reactive cysteine residue (Cys227) in human carbonylreductase

Carbonyl reductase is highly susceptible to inactivation by organomercurial s suggesting the presence of a reactive cysteine residue in, or close to, t he active site. This residue is also close to a site which binds glutathion e. Structurally, carbonyl reductase belongs to the short-chain dehydrogenas e/reductase family and contains five cysteine residues, none of which is co nserved within the family. In order to identify the reactive residue and in vestigate its possible role in glutathione binding, alanine was substituted for each cysteine residue of human carbonyl reductase by site-directed mut agenesis. The mutant enzymes were expressed in Escherichia coli and purifie d to homogeneity. Four of the five mutants (C26A, C122A C150A. and C226A) e xhibited wild-type-like enzyme activity, although K-m values of C226A for t hree structurally different substrates were increased threefold to 10-fold. The fifth mutant, C227A, showed a 10-15-fold decrease in k(cat) and a thre efold to 40-fold increase in K-m, resulting in a 30-500-fold drop in k(cat) /K-m. NaCl (300 mM) increased the activity of C227A 16-fold, whereas the ac tivity of the wild-type enzyme was only doubled. Substitution of serine rat her than alanine for Cys227 similarly affected the kinetic constants with t he exception that NaCl did not activate the enzyme. Both C227A and C227S mu tants were insensitive to inactivation by 4-hydroxymercuribenzoate. Unlike the parent carbonyl compounds, the glutathione adducts of menadione and pro staglandin Al were better substrates for the C227A and C227S mutants than t he wild-type enzyme. Conversely, the binding of free glutathione to both mu tants was reduced. Our findings indicate that Cys227 is the reactive residu e and suggest that it is involved in the binding of both substrate and glut athione.