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
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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DOI：
10.1042/bj20050167
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发表时间：
2005-07-15
影响因子：
4.1
通讯作者：
Nidetzky, B
中科院分区：
文献类型：
--
作者：
Kratzer, R;Nidetzky, B
细假丝酵母木糖还原酶（AKR 2B 5）的Lys-80在醛酮还原酶蛋白超家族中是保守的，并且可以引发附近的Tyr-51用于一般酸催化NAD（P）H依赖性羰基还原。我们已经研究了两个AKR 2B 5突变体，Lys-80 -> Ala（K80 A）和Asp-46 ->阿斯蒂Lys-80 -> Ala（D46 N K80 A）中侧链取代的催化意义，使用稳态动力学分析和用外部胺恢复活性。K80 A中NAD（+）（Kd = 24 μ M）和NADP（+）（Kd = 0.03 μ M）的结合比野生型酶紧密10倍和40倍，而在该突变体中NADH（Kd = 51 AM）和NADPH（Kd = 19 AM）的结合分别减弱2倍和16倍。D46 N K80 A约均匀地结合NAD（P）H和NAD（P）（+）。5-与野生型酶相比，折叠不那么紧密。质子化乙胺非共价还原依赖于NADH的还原酶活性的二级速率常数（k（max）/K-胺）为0.11 M-1（.）s（-1），而D46 N K80 A没有检测到拯救。在校正侧链疏水性的影响后，我们获得了log（k（max）/K-胺）和胺基pK（a）的线性自由能关系（斜率=+0.29; r（2）= 0.93），pH为7.0。野生型和D46 N K80 A羰基还原的log（k（cat）/K-m）pH曲线显示相同且动力学未扰动的pKa值为8.50（+/- 0.20）。因此，Lys-80的质子化侧链不是AKR 2B 5的一般酸催化的必要活化剂。通过与带负电荷的Asp-46的盐连接相互作用在结构上稳定，建议将Tyr-51的侧链拉到催化位置，导致整体中性电荷的预组织极性环境，其中不带电荷的反应基团的近似是有利的，因此强烈优选从NAD（P）H的氢化物转移。与NAD（P）（+）结合选择性相比，Lys-80通过增加NAD（P）H进一步影响AKR 2B 5对NAD（P）H依赖性还原的方向偏好。
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 -> Asti Lys-80 -> Ala (D46N K80A), using steady-state kinetic analysis and restoration of activity with external amines. Binding of NAD(+) (K-d = 24 mu M) and NADP(+) (K-d = 0.03 mu M) was 10- and 40-fold tighter in K80A than the wild-type enzyme, whereas binding of NADH (K-d = 51 AM) and NADPH (K-d = 19 AM) 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 (k(max)/K-amine) 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 (k(max)/K-amine) and amine group pK(a) (slope = + 0.29; r(2) = 0.93) at pH 7.0. pH profiles of log (k(cat)/K-m) 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.