Multiple forms of xylose reductase in Candida intermedia: comparison of their functional properties using quantitative structure-activity relationships, steady-state kinetic analysis, and pH studies.
The xylose-fermenting yeast Candida intermedia produces two isoforms of xylose reductase: one is NADPH-dependent (monospecific xylose reductase; msXR), and another is shown here to prefer NADH approximately 4-fold over NADPH (dual specific xylose reductase; dsXR). To compare the functional properties of the isozymes, a steady-state kinetic analysis for the reaction d-xylose + NAD(P)H + H(+) <--> xylitol + NAD(P)(+) was carried out and specificity constants (k(cat)/K(aldehyde)) were measured for the reduction of a series of aldehydes differing in side-chain size as well as hydrogen-bonding capabilities with the substrate binding pocket of the enzyme. dsXR binds NAD(P)(+) (K(iNAD+) = 70 microM; K(iNADP+) = 55 microM) weakly and NADH (K(i) = 8 microM) about as tightly as NADPH (K(i) = 14 microM). msXR shows uniform binding of NADPH and NADP(+) (K(iNADP+) approximately K(iNADPH) = 20 microM). A quantitative structure-activity relationship analysis was carried out by correlating logarithmic k(cat)/K(aldehyde) values for dsXR with corresponding logarithmic k(cat)/K(aldehyde) values for msXR. This correlation is linear with a slope of approximately 1 (r (2) = 0.912), indicating that no isozyme-related pattern of substrate specificity prevails and aldehyde-binding modes are identical in both XR forms. Binary complexes of dsXR-NADH and msXR-NADPH show the same macroscopic pK of approximately 9.0-9.5, above which the activity is lost in both enzymes. A lower pK of 7.4 is seen for dsXR-NADPH. Specificity for NADH and greater binding affinity for NAD(P)H than NAD(P)(+) are thus the main features of enzymic function that distinguish dsXR from msXR.
Nidetzky
Bernd
B
Institute of Biotechnology, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria. bernd.nidetzky@tugraz.at
Brüggler
Kaspar
K
Kratzer
Regina
R
Mayr
Peter
P
D000449:Aldehyde Reductase / Q000737:chemistry* / Q000378:metabolism*; D002175:Candida / Q000201:enzymology*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D021281:Quantitative Structure-Activity Relationship; D013816:Thermodynamics; D014994:Xylose / Q000378:metabolism
D007527:Isoenzymes; D009249:NADP; D014994:Xylose; D000449:Aldehyde Reductase