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>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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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
pLys-80 of iCandida tenuis/i 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 NADsup+/sup (iK/isubd/sub=24 μM) and NADPsup+/sup (iK/isubd/sub=0.03 μM) was 10- and 40-fold tighter in K80A than the wild-type enzyme, whereas binding of NADH (iK/isubd/sub=51 μM) and NADPH (iK/isubd/sub=19 μM) was weakened 2- and 16-fold in this mutant respectively. D46N K80A bound NAD(P)H and NAD(P)sup+/sup 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 (ik/isubmax/sub/iK/isubamine/sub) by protonated ethylamine was 0.11 Msup?1/sup·ssup?1/sup 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 (ik/isubmax/sub/iK/isubamine/sub) and amine group piK/isuba/sub (slope=+0.29; ir/isup2/sup=0.93) at pH 7.0. pH profiles of log (ik/isubcat/sub/iK/isubm/sub) for carbonyl group reduction by wild-type and D46N K80A revealed identical and kinetically unperturbed piK/isuba/sub 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)sup+/sup-binding selectivity./p
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机译：> Candida tenuis 木糖还原酶（AKR2B5）的Lys-80在整个aldo-keto还原酶蛋白超家族中都是保守的，并且可能引发附近的Tyr-51进行一般的酸催化，形成NAD（P）H-依赖的羰基还原。我们使用稳态动力学分析和活性恢复，研究了两个AKR2B5突变体Lys-80→Ala（K80A）和Asp-46→Asn Lys-80→Ala（D46N K80A）侧链取代的催化意义。与外部胺。 NAD + （ K d =24μM）和NADP + （ K d = 0.03μM）在K80A中比野生型酶紧10到40倍，而NADH（ K d =51μM）和NADPH（ K d =19μM）在该突变体中分别被削弱了2倍和16倍。 D46N K80A与NAD（P）H和NAD（P） + 结合的均匀性比野生型酶低约5倍。非共价还原NADH依赖的还原酶活性（ k max / K 胺）对于K80A，质子化的乙胺为0.11M ？1 ·s ？1 ，而对于D46N K80A，没有可检测到的拯救。在校正了侧链疏水性的影响后，我们获得了log（ k max / K 胺< / sub>）和胺基p K a （slope = + 0.29; r 2 = 0.93） pH值7.0。通过野生型和D46N K80A还原羰基的log（ k cat / K m ）的pH谱揭示了相同且在动力学上不受干扰的p K a 值为8.50（±0.20）。因此，Lys-80的质子化侧链不是AKR2B5催化一般酸的必要活化剂。通过与带负电荷的Asp-46的盐键相互作用在结构上稳定，建议将Tyr-51的侧链拉到催化位置，从而导致整体中性电荷的预先组织化极性环境，在该环境中近似不带电荷的反应性基团是有利的，因此强烈优选从NAD（P）H转移氢化物。与NAD（P） + -结合选择性相比，Lys-80通过增加NAD（P）H进一步影响AKR2B5对NAD（P）H依赖性还原的方向性偏好。
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