Evidence in Support of Lysine 77 and Histidine 96 as Acid-Base Catalytic Residues in Saccharopine Dehydrogenase from Saccharomyces cerevisiae
Evidence in Support of Lysine 77 and Histidine 96 as Acid-Base Catalytic Residues in Saccharopine Dehydrogenase from Saccharomyces cerevisiae
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DOI：
10.1021/bi201808
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发表时间：
2012-01-31
期刊：
影响因子：
2.9
通讯作者：
West, Ann H.
中科院分区：
文献类型：
--
作者：
Kumar, Vidya Prasanna;Thomas, Leonard M.;West, Ann H.
蔗糖苷脱氢酶是以NAD(+)为氧化剂，催化已二酸-α-氨基途径的最终反应，即L-糖苷转化为L-赖氨酸(Lys)和α-酮戊二酸(α-Kg)。该酶利用一般的酸碱机理与一个碱基和一个基团进行反应，该碱基被认为在氧化步骤中接受来自糖胺的仲胺的质子，另一个基团被提议活化水来水解亚胺。分别在2.0埃和2.2埃分辨率下测定了该酶与糖胺和NADH结合的开放形式和封闭形式的晶体结构。在三元络合物中，结构域I相对于结构域II发生了显著的移动，关闭了两个结构域之间的活性部位裂隙，并使H96和K77接近底物结合部位。H96和K77的氢化物转移距离为3.6埃，H96和K77的侧链位置合适，可作为酸碱催化剂。K77M和H96Q单突变和K77M/H96Q双突变酶为SDH反应提供了通用的酸碱催化剂。K77的侧链最初接受来自底物Lys的e-胺的质子，并最终在氢化物转移步骤中将其还原为仲胺时将其提供给亚氨基氮，而H96在生成甲醇胺时质子化羰基氧。K77M、H976Q和K77M/H96Q突变酶使V/E-t和V-2/KLysEt和V-2/Kα-KGET分别降低145倍、28倍和700倍，V-2/KLysEt和V-2/Kα-KGET增加10(3)倍(双突变使>；二阶速率常数降低10(5)倍)。此外，K77M突变酶的初级动力学同位素效应为2.0，逆溶剂同位素效应为0.77onV(2)/K-Lys。当在D2O中重复主要的动力学同位素效应时，V-D(2)/K-Lys)(D1O)的值也是2.0，这与限速氢化物转移步骤一致。在V-2/K-Lys上观察到0.8的粘度效应，这表明在氢化物转移之前酶形式的稳定是溶剂氢同位素效应的结果。在V上观察到一个小的正常溶剂同位素效应，当重复使用NADD时，该效应略有减小，这与产物释放对速率限制的贡献一致。此外，V-2/KLysEt不依赖于pH，这与失去酸碱催化剂和第二催化基团的pK(A)微扰到更高的pH是一致的，这可能是由于活性中心的总电荷改变的结果。H96Q在H2O或D2O中测量的初级动力学同位素效应的误差在1以内。以NADH或NADD为二核苷酸底物时，观测到的溶剂氚同位素效应为2.4。数据表明亚胺的形成是限速的，这与H96在亚胺形成时质子化离开羟基的作用是一致的。V-2/KLysEt的pH速率曲线显示K77的pKa，微扰到类似于9的值，必须去质子化才能接受来自底物Lys的异构胺的质子，以便它能够起到亲核剂的作用。总体而言，数据与K77作为碱的作用是一致的，该碱在α-酮戊二酸的α-氧基攻击底物赖氨酸的异构胺之前接受来自底物赖氨酸的异构胺的质子，最后给亚胺氮一个质子，因为它被还原为糖胺。此外，数据表明，H96在赖氨酸的e-胺和α-酮戊二酸的α-氧基之间形成亚胺的酸碱催化剂中起到了作用。
Saccharopine dehydrogenase (SDH) catalyzes the final reaction in the alpha-aminoadipate pathway, the conversion of L-saccharopine to L-lysine (Lys) and alpha-ketoglutarate (alpha-kg) using NAD(+) as an oxidant. The enzyme utilizes a general acid-base mechanism to conduct its reaction with a base proposed to accept a proton from the secondary amine of saccharopine in the oxidation step and a group proposed to activate water to hydrolyze the resulting imine. Crystal structures of an open apo form and a closed form of the enzyme with saccharopine and NADH bound have been determined at 2.0 and 2.2 angstrom resolution, respectively. In the ternary complex, a significant movement of domain I relative to domain II that closes the active site cleft between the two domains and brings H96 and K77 into the proximity of the substrate binding site is observed. The hydride transfer distance is 3.6 angstrom, and the side chains of H96 and K77 are properly positioned to act as acid-base catalysts. K77M and H96Q single-mutant and K77M/H96Q double-mutant enzymes provides general acid-base catalysts in the SDH reaction. The side chain of K77 initially accepts a proton from the e-amine of the substrate Lys and eventually donates it to the imino nitrogen as it is reduced to a secondary amine in the hydride transfer step, and H96 protonates the carbonyl oxygen as the carbinolamine is formed. The K77M, H976Q, and K77M/H96Q mutant enzymes give 145-, 28-, and 700-fold decreases in V/E-t and >10(3)-fold increases in V-2/KLysEt and V-2/K alpha-kgEt (the double mutation gives >10(5)-fold decreases in the second-order rate constants). In addition, the K77M mutant enzyme exhibits a primary deuterium kinetic isotope effect of 2.0 and an inverse solvent deuterium isotope effect of 0.77 onV(2)/K-Lys. A value of 2.0 was also observed for V-D(2)/K-Lys)(D1O) when the primary deuterium kinetic isotope effect was repeated in D2O, consistent with a rate-limiting hydride transfer step. A viscosity effect of 0.8 was observed on V-2/K-Lys, indicating the solvent deuterium isotope effect resulted from stabilization of an enzyme form prior to hydride transfer. A small normal solvent isotope effect is observed on V, which decreases slightly when repeated with NADD, consistent with a contribution from product release to rate limitation. In addition, V-2/KLysEt is pH-independent, which is consistent with the loss of an acid base catalyst and perturbation of the pK(a) of the second catalytic group to a higher pH, likely a result of a change in the overall charge of the active site. The primary deuterium kinetic isotope effect for H96Q, measured in H2O or D2O, is within error equal to 1. A solvent deuterium isotope effect of 2.4 is observed with NADH or NADD as the dinucleotide substrate. Data suggest rate-limiting imine formation, consistent with the proposed role of H96 in protonating the leaving hydroxyl as the imine is formed. The pH rate profile for V-2/KLysEt exhibits the pKa for K77, perturbed to a value of similar to 9, which must be unprotonated to accept a proton from the epsilon-amine of the substrate Lys so that it can act as a nucleophile.Overall, data are consistent with a role for K77 acting as the base that accepts a proton from the epsilon-amine of the substrate lysine prior to nucleopilic attack on the alpha-oxo group of alpha-ketoglutarate, and finally donating a proton to the imine nitrogen as it is reduced to give saccharopine. In addition, data indicate a role for H96 acting as a general acid-base catalyst in the formation of the imine between the e-amine of lysine and the alpha-oxo group of alpha-ketoglutarate.