Probing the affinity and specificity of yeast alcohol dehydrogenase I for coenzymes

Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (SceADH) binds NAD (+) and NADH less tightly and turns over substrates more rapidly than does horse (Equus caballus) liver alcohol dehydrogenase E isoenzyme (EcaADH), an d neither enzyme uses NADP efficiently. Amino acid residues in the proposed adenylate binding pocket of SceADH were substituted in attempts to improve affinity for coenzymes or reactivity with NADP, Substitutions in SceADH (G ly202Ile or Ser246Ile) with the corresponding residues in the adenine bindi ng site of the homologous EcaADH have modest effects on coenzyme binding an d other kinetic constants, but the Ser246Ile substitution decreases turnove r numbers by 350-fold. The Ser176Phe substitution (also near adenine site) significantly decreases affinity for coenzymes and turnover numbers, In the consensus nucleotide-binding beta alpha beta fold sequence, SceADH has two alanine residues (177-G<(AA)under bar>GGLG-183) instead of the Leu200 in E caADH (199-G (L) under bar GGVG-204); the Ala178-Ala179 to Leu substitution significantly decreases affinity for coenzymes and turnover numbers. Some NADP-dependent enzymes have an Ala corresponding to Gly183 in SceADH; the G ly183Ala substitution significantly decreases affinity for coenzymes and tu rnover numbers. NADP-dependent enzymes usually have a neutral residue inste ad of the Asp (Asp201 in SceADH) that interacts with the hydroxyl groups of the adenosine ribose, along with a basic residue (at position 202 or 203) to stabilize the S'-phosphate of NADP, The Gly203Arg change in SceADH does not significantly affect the kinetics. The Gly183Ala or Gly203Arg substitut ions do not enable SceADH to use NADP(+) as coenzyme, SceADH with the singl e Asp201Gly or double Asp201Gly:Gly203Arg substitutions have similar, low a ctivity with NADP(+), The results suggest that several of the amino acid re sidues participate in coenzyme binding and that conversion of specificity f or coenzyme requires multiple substitutions. (C) 1999 Academic Press.