Roles of tyrosine 158 and lysine 165 in the catalytic mechanism of InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis
Roles of tyrosine 158 and lysine 165 in the catalytic mechanism of InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis
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DOI：
10.1021/bi990529c
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发表时间：
1999-10-12
期刊：
影响因子：
2.9
通讯作者：
Tonge, PJ
中科院分区：
文献类型：
--
作者：
Parikh, S;Moynihan, DP;Tonge, PJ
研究了酪氨酸158（Y158）和赖氨酸165（K165）在结核分枝杆菌烯酰ACP还原酶InhA催化机制中的作用。基于与短链醇脱氢酶家族的结构和序列同源性，这些残基已被鉴定为推定的催化残基。用苯丙氨酸（Y158 F）和丙氨酸（Y158 A）替换Y158分别导致k（cat）降低24倍和1500倍，而使底物反式-2-十二烯酰-CoA的Km不受影响。然而，值得注意的是，用丝氨酸（Y158 S）替换Y158导致具有野生型活性的酶。动力学同位素效应研究表明，溶剂可交换质子的转移是部分限速的野生型和Y158 S酶，但不是Y158 A酶。这些数据表明，Y158在反应中不起质子供体的作用，但可能起亲电催化剂的作用，通过与底物羰基的氢键合稳定氢化物转移的过渡态。一个构象的变化，涉及旋转的Y158侧链的烯酰底物结合酶提出作为一个解释V/KDD-CoA上观察到的逆溶剂同位素效应时，无论是NADH或NADD被用作还原剂。这些数据与最近公布的与InhA结合的C16脂肪酸底物的结构一致，其显示Y158氢键合至底物羰基并从其在InhA-NADH二元复合物中占据的位置旋转[Rozwarski，D.一、维尔切兹角Sugantino，M.，比特曼河，和Sacchettini，J. C.（1999）J.Biol.Chem.274，15582-15589]。最后，使用定点突变分析了K165的作用。用谷氨酰胺（K165 Q）和精氨酸（K165 R）取代K165对酶的催化能力或结合NADH的能力没有影响。然而，K165 A和K165 M酶不能结合NADH，表明K165在辅因子结合中具有主要作用。
The role of tyrosine 158 (Y158) and lysine 165 (K165) in the catalytic mechanism of InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis, has been investigated. These residues have been identified as putative catalytic residues on the basis of structural and sequence homology with the short chain alcohol dehydrogenase family of enzymes. Replacement of Y158 with phenylalanine (Y158F) and with alanine (Y158A) results in 24- and 1500-fold decreases in k(cat), respectively, while leaving K-m for the substrate, trans-2-dodecenoyl-CoA, unaffected. Remarkably, however, replacement of Y158 with serine (Y158S) results in an enzyme with wild-type activity. Kinetic isotope effect studies indicate that the transfer of a solvent-exchangeable proton is partially rate-limiting for the wild-type and Y158S enzymes, but not for the Y158A enzyme. These data indicate that Y158 does not function formally as a proton donor in the reaction but likely functions as an electrophilic catalyst, stabilizing the transition state for hydride transfer by hydrogen bonding to the substrate carbonyl. A conformational change involving rotation of the Y158 side chain upon binding of the enoyl substrate to the enzyme is proposed as an explanation for the inverse solvent isotope effect observed on V/KDD-CoA when either NADH or NADD is used as the reductant. These data are consistent with the recently published structure of a C16 fatty acid substrate bound to InhA that shows Y158 hydrogen bonded to the substrate carbonyl group and rotated from the position it occupies in the InhA-NADH binary complex [Rozwarski, D. A., Vilcheze, C., Sugantino, M., Bittman, R., and Sacchettini, J. C. (1999) J. Biol. Chem. 274, 15582-15589]. Finally, the role of K165 has been analyzed using site-directed mutagenesis. Replacement of K165 with glutamine (K165Q) and arginine (K165R) has no effect on the enzyme's catalytic ability or on its ability to bind NADH. However, the K165A and K165M enzymes are unable to bind NADH, indicating that K165 has a primary role in cofactor binding.