NAD(P)H-DEPENDENT ALDOSE REDUCTASE FROM THE XYLOSE-ASSIMILATING YEASTCANDIDA-TENUIS - ISOLATION, CHARACTERIZATION AND BIOCHEMICAL PROPERTIES OF THE ENZYME

During growth on D-xylose the yeast Candida tenuis produces one aldose reductase that is active with both NADPH and NADH as coenzyme. This e nzyme has been isolated by dye ligand and anion-exchange chromatograph y in yields of 76 qb. Aldose reductase consists of a single 43 kDa pol ypeptide with an isoelectric point of 4.70. Initial velocity, product inhibition and binding studies are consistent with a compulsory-ordere d, ternary-complex mechanism with coenzyme binding first and leaving l ast. The catalytic efficiency (k(cat)/K-m) in D-xylose reduction at pH 7 is more than 60-fold higher than that in xylitol oxidation and refl ects significant differences in the corresponding catalytic centre act ivities as well as apparent substrate-binding constants. The enzyme pr efers NADP(H) approx. 2-fold to NAD(H), which is Largely due to better apparent binding of the phosphorylated form of the coenzyme. NADP(+) is a potent competitive inhibitor of the NADH-linked aldehyde reductio n (K-1 1.5 mu M), whereas NAD(+) is not. Unlike mammalian aldose reduc tase, the enzyme from C. tenuis is not subject to oxidation-induced ac tivation. Evidence of an essential lysine residue located in or near t he coenzyme binding site has been obtained from chemical modification of aldose reductase with pyridoxal 5'-phosphate. The results are discu ssed in the context of a comparison of the enzymic properties of yeast and mammalian aldose reductase.