Identification of the reactive cysteine residue (Cys227) in human carbonyl reductase

Abstract

Carbonyl reductase is highly susceptible to inactivation by organomercurials suggesting the presence of a reactive cysteine residue in, or close to, the active site. This residue is also close to a site which binds glutathione. Structurally, carbonyl reductase belongs to the short-chain dehydrogenase/reductase family and contains five cysteine residues, none of which is conserved within the family. In order to identify the reactive residue and investigate its possible role in glutathione binding, alanine was substituted for each cysteine residue of human carbonyl reductase by site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli and purified to homogeneity. Four of the five mutants (C26A, C122A C150A and C226A) exhibited wild-type-like enzyme activity, although K m values of C226A for three structurally different substrates were increased threefold to 10-fold. The fifth mutant, C227A, showed a 10±15-fold decrease in k cat and a threefold 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 activity of the wild-type enzyme was only doubled. Substitution of serine rather than alanine for Cys227 similarly affected the kinetic constants with the exception that NaCl did not activate the enzyme. Both C227A and C227S mutants were insensitive to inactivation by 4-hydroxymercuribenzoate. Unlike the parent carbonyl compounds, the glutathione adducts of menadione and prostaglandin A 1 were better substrates for the C227A and C227S mutants than the wild-type enzyme. Conversely, the binding of free glutathione to both mutants was reduced. Our findings indicate that Cys227 is the reactive residue and suggest that it is involved in the binding of both substrate and glutathione.

Human Carbonyl Reductase 1 Is an S-Nitrosoglutathione Reductase

“…One well accepted aspect of hCBR1 substrate recognition is the presence of a GSH-binding pocket predicted to be in close proximity to the catalytic site. GSH conjugates of otherwise poorly recognized substrates such as prostaglandin A1 are reduced by hCBR1 (4), supporting this hypothesis. To identify additional physiological substrates of hCBR1, we initiated a structural biology effort to analyze the GSH-binding site of hCBR1, hypothesizing that cellular GSH adducts might serve as particularly good hCBR1 substrates.…”

“…S1) as previously described (3). The crystals were soaked successively (three times) in precipitant solution containing 5 mM of either freshly prepared HMGSH 4 or GSH. Crystals for hCBR1⅐NADP were grown from 20% polyethylene glycol 3350 and 0.2 M NaCl in space group P4 3 2 1 2 with one molecule in the asymmetric unit.…”

“…Determination of NADPH and GSNO Stoichiometry-Reactions were prepared in the manner described for K m determination except substrate concentrations including 100 M NADPH and 50 M GSNO, 50 M NADPH and 100 M GSNO, 4 HMGSH was produced in situ by incubating stoichiometric amounts of GSH and formaldehyde and yielded the desired linear conjugate rather than the previously found bicyclic adduct ((2S,7R)-7-(carboxymethylcarbamoyl)-5-oxo-9-thia-1,6-diaza-,bicyclo[4.4.1]undecane-2-carboxylic acid) (5). or 50 M NADPH and 50 M GSNO.…”

Inhibition of Anthracycline Alcohol Metabolite Formation in Human Heart Cytosol: A Potential Role for Several Promising Drugs

“…CBR1 primarily reduces DNR to DNRol, whereas AKR1A1 prevalently converts DOX into DOXol (Mordente et al, 2003;Salvatorelli et al, 2007;Kassner et al, 2008). Human CBR1 contains five cysteines located in, or close to, the active site, and among them Cys227 has been identified as the residue involved in the binding of both substrate and GSH (Tinguely and Wermuth, 1999;Hartmanová et al, 2013). AKR1A1 contains six cysteines, none of which appears to be implicated in the catalytic mechanism of the enzyme (Barski et al, 2008).…”

Characterization of the Canine Anthracycline-Metabolizing Enzyme Carbonyl Reductase 1 (cbr1) and the Functional Isoform cbr1 V218

“…5). A cysteine residue in human CBR1 observed at position 227 previously identified to be important for catalysis of some nonglutathione-bound substrate binding is also observed in the canine protein sequence (Tinguely and Wermuth, 1999). The close proximity of the V218 variant to critical active-site amino acid residues, and change from an acidic (aspartic acid) to nonpolar (valine) residue could then be hypothesized to influence the binding interaction between daunorubicin and catalytic residues within the binding pocket of canine cbr1.…”