Electrostatic stabilization in a pre-organized polar active site: the catalytic role of Lys-80 in Candida tenuis xylose reductase (AKR2B5) probed by site-directed mutagenesis and functional complementation studies.
Lys-80 of Candida tenuis xylose reductase (AKR2B5) is conserved throughout the aldo-keto reductase protein superfamily and may prime the nearby Tyr-51 for general acid catalysis to NAD(P)H-dependent carbonyl group reduction. We have examined the catalytic significance of side-chain substitutions in two AKR2B5 mutants, Lys-80-->Ala (K80A) and Asp-46-->Asn Lys-80-->Ala (D46N K80A), using steady-state kinetic analysis and restoration of activity with external amines. Binding of NAD+ (Kd = 24 microM) and NADP+ (Kd = 0.03 microM) was 10- and 40-fold tighter in K80A than the wild-type enzyme, whereas binding of NADH (Kd = 51 microM) and NADPH (Kd = 19 microM) was weakened 2- and 16-fold in this mutant respectively. D46N K80A bound NAD(P)H and NAD(P)+ uniformly approx. 5-fold less tightly than the wild-type enzyme. The second-order rate constant for non-covalent restoration of NADH-dependent reductase activity (kmax/Kamine) by protonated ethylamine was 0.11 M(-1).s(-1) for K80A, whereas no detectable rescue occurred for D46N K80A. After correction for effects of side-chain hydrophobicity, we obtained a linear free energy relationship of log (kmax/Kamine) and amine group pKa (slope = +0.29; r2 = 0.93) at pH 7.0. pH profiles of log (kcat/Km) for carbonyl group reduction by wild-type and D46N K80A revealed identical and kinetically unperturbed pKa values of 8.50 (+/-0.20). Therefore the protonated side chain of Lys-80 is not an essential activator of general acid catalysis by AKR2B5. Stabilized structurally through the salt-link interaction with the negatively charged Asp-46, it is proposed to pull the side chain of Tyr-51 into the catalytic position, leading to a preorganized polar environment of overall neutral charge, in which approximation of uncharged reactive groups is favoured and thus hydride transfer from NAD(P)H is strongly preferred. Lys-80 affects further the directional preference of AKR2B5 for NAD(P)H-dependent reduction by increasing NAD(P)H compared with NAD(P)+-binding selectivity.
Kratzer
Regina
R
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria.
Nidetzky
Bernd
B
D000449:Aldehyde Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010293:Pargyline / Q000031:analogs & derivatives / Q000494:pharmacology; D011437:Propylamines / Q000494:pharmacology; D011485:Protein Binding; D055672:Static Electricity; D013379:Substrate Specificity; D014993:Xylitol / Q000378:metabolism; D014994:Xylose / Q000378:metabolism
D011437:Propylamines; C009054:propargylamine; D009249:NADP; D010293:Pargyline; D014994:Xylose; D000449:Aldehyde Reductase; D014993:Xylitol