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 μM) and NADP+ (Kd=0.03 μM) was 10- and 40-fold tighter in K80A than the wild-type enzyme, whereas binding of NADH (Kd=51 μM) and NADPH (Kd=19 μM) 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.
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
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Regina Kratzer, Bernd Nidetzky; 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. Biochem J 15 July 2005; 389 (2): 507–515. doi: https://doi.org/10.1042/BJ20050167
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