Roles of His-79 and Tyr-180 of D-xylose/dihydrodiol dehydrogenase in catalytic function.
Mammalian dimeric dihydrodiol dehydrogenase is identical with d-xylose dehydrogenase and belongs to a protein family with prokaryotic proteins including glucose-fructose oxidoreductase. Of the conserved residues in this family, either His-79 or Tyr-180 of d-xylose/dihydrodiol dehydrogenase has been proposed to be involved in the catalytic function. Site-directed mutagenesis was used to examine the roles of the two residues of the monkey enzyme. A mutant, Y180F, was almost inactive, but, similarly to the wild-type enzyme, exhibited high affinity for NADP(H) and fluorescence energy transfer upon binding of NADPH. The H79Q mutation had kinetically largest effects on K(d) (>7-fold increase) and K(m) (>25-fold increase) for NADP(H), and eliminated the fluorescence energy transfer. Interestingly, the dehydrogenase activity of this mutant was potently inhibited with a 190-fold increase in the K(m) for NADP(+) by high ionic strength, which activated the activity of the wild-type enzyme. These results suggest a critical role of Tyr-180 in the catalytic function of this class of enzymes, in addition to functions of His-79 in the coenzyme binding and chemical steps of the reaction.
Asada
Y
Y
Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu, Japan.
Aoki
S
S
Ishikura
S
S
Usami
N
N
Hara
A
A
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D000882:Haplorhini; D006639:Histidine / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D014994:Xylose / Q000378:metabolism*
D017931:DNA Primers; D014443:Tyrosine; D006639:Histidine; D014994:Xylose; D010088:Oxidoreductases; C048338:trans-1,2-dihydrobenzene-1,2-diol dehydrogenase