Parikh S, Moynihan D P, Xiao G, Tonge P J
Department of Chemistry, State University of New York at Stony Brook 11794-3400, USA.
Biochemistry. 1999 Oct 12;38(41):13623-34. doi: 10.1021/bi990529c.
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/K(DD-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.
已对结核分枝杆菌烯酰 - ACP还原酶InhA催化机制中酪氨酸158（Y158）和赖氨酸165（K165）的作用进行了研究。基于与短链醇脱氢酶家族酶的结构和序列同源性，这些残基已被确定为推定的催化残基。用苯丙氨酸（Y158F）和丙氨酸（Y158A）取代Y158分别导致催化常数（k(cat)）降低24倍和1500倍，而底物反式 - 2 - 十二碳烯酰 - CoA的米氏常数（K(m)）不受影响。然而，值得注意的是，用丝氨酸（Y158S）取代Y158会产生具有野生型活性的酶。动力学同位素效应研究表明，对于野生型和Y158S酶，溶剂可交换质子的转移部分限制了反应速率，但对于Y158A酶则不然。这些数据表明，Y158在反应中并非正式地作为质子供体起作用，而可能作为亲电催化剂，通过与底物羰基形成氢键来稳定氢化物转移的过渡态。有人提出，当使用NADH或NADD作为还原剂时，烯酰底物与酶结合后Y158侧链的旋转所涉及的构象变化可解释在V/K(DD - CoA)上观察到的反向溶剂同位素效应。这些数据与最近发表的与InhA结合的C16脂肪酸底物的结构一致，该结构显示Y158与底物羰基形成氢键并从其在InhA - NADH二元复合物中占据的位置旋转[罗兹瓦尔基，D. A.，维尔切泽，C.，苏甘蒂诺，M.，比特曼，R.，和萨切蒂尼，J. C.（1999年）《生物化学杂志》274，15582 - 15589]。最后，使用定点诱变分析了K165的作用。用谷氨酰胺（K165Q）和精氨酸（K165R）取代K165对酶的催化能力或其结合NADH的能力没有影响。然而，K165A和K165M酶无法结合NADH，表明K165在辅因子结合中起主要作用。
