Coenzyme specificity of human monomeric carbonyl reductase: contribution of Lys-15, Ala-37 and Arg-38.
Short-chain dehydrogenases/reductases catalyze the oxidoreduction of alcohol and carbonyl compounds using either NAD or NADPH as coenzyme. Structural analysis suggests that specificity for NADPH is conferred by two highly conserved basic residues in the N-terminal part of the peptide chain, whereas specificity for NAD correlates with the presence of an Asp adjacent to the position of the distal basic residue in NADP-dependent enzymes. We carried out site-directed mutagenesis of the two basic residues: Lys-15 and Arg-38, as well as of Ala-37 of human monomeric carbonyl reductase in order to investigate their contribution to coenzyme binding and specificity. Substitution of Lys-15 or Arg-38 by Gln and, even more pronounced Asp decreased the catalytic efficiency (k(cat)/K(m,NADPH)) by more than three orders of magnitude. Similarly, substitution of Asp for Ala-37 decreased k(cat)/K(m,NADPH) 1000-fold but had little effect on k(cat)/K(m,NADH). The results demonstrate the importance of basic residues at positions 15 and 38 and the absence of an acidic residue at position 37 for NADPH binding and catalysis.
Sciotti
M
M
Central Chemical Laboratory of the University Clinics, Inselspital, CH-3010, Bern, Switzerland.
Wermuth
B
B
D000409:Alanine / Q000737:chemistry; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D003067:Coenzymes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D003067:Coenzymes; D011994:Recombinant Proteins; D009243:NAD; D009249:NADP; D001120:Arginine; D000429:Alcohol Oxidoreductases; D008239:Lysine; D000409:Alanine