THE FUNCTION OF AMINO-ACID-RESIDUES CONTACTING THE NICOTINAMIDE RING OF NADPH IN DIHYDROFOLATE-REDUCTASE FROM ESCHERICHIA-COLI
THE FUNCTION OF AMINO-ACID-RESIDUES CONTACTING THE NICOTINAMIDE RING OF NADPH IN DIHYDROFOLATE-REDUCTASE FROM ESCHERICHIA-COLI
复制标题
DOI：
10.1021/bi00110a006
复制
发表时间：
1991-11-19
期刊：
影响因子：
2.9
通讯作者：
BENKOVIC, SJ
中科院分区：
文献类型：
--
作者：
ADAMS, JA;FIERKE, CA;BENKOVIC, SJ
通过定点诱变和详细的稳态和预稳态动力学实验，确定了大肠杆菌二氢叶酸还原酶中与NADPH烟酰胺环接触的三个氨基酸残基的重要性。用甘氨酸或异亮氨酸（Y100G或Y100I）替代Tyr-100会破坏酚侧链和烟酰胺环之间的芳香-芳香相互作用。这两种突变都去除了氧化和还原辅酶的差异结合，这意味着tyr1 -100是辅酶特异性的主要决定因素。将Ser-49替换为丙氨酸（S49A），旨在取代或降低接触烟酰胺环N1的结合水分子的极化率，仅影响NADP+的释放速度。用丙氨酸（I14A）替代Ile-14，旨在改变与烟酰胺环外围的弱极性和氢键相互作用，仅影响NADPH的结合。Y100I， y10og和I14A都使化学步骤的激活势垒增加了大约2千卡/摩尔。对S49A没有影响，说明水结构对稳定氢化物转移过渡态并不重要。此外，观察到的这些突变的名义效应不支持邻近氨基酸残基通过极性或弱极性接触参与反应过渡态稳定的假设。
The importance of three amino acid residues contacting the nicotinamide ring of NADPH in Escherichia coli dihydrofolate reductase has been defined using site-directed mutagenesis and detailed steady-state and pre-steady-state kinetic experiments. Replacement of Tyr-100 with either glycine or isoleucine (Y100G or Y100I) disrupts an aromatic-aromatic interaction between the phenolic side chain and the nicotinamide ring. Both mutations remove the differential binding of the oxidized and reduced coenzymes implicating Tyr-100 as a major determinant for coenzyme specificity. Replacement of Ser-49 for alanine (S49A), designed to either displace or reduce the polarizability of a bound water molecule contacting the N1 of the nicotinamide ring, affects only the rate of release of NADP+. Replacement of Ile-14 with alanine (I14A), designed to alter both a weakly polar and a hydrogen bonding interaction with the periphery of the nicotinamide ring, affects only the binding of NADPH. Y100I, Y1OOG, and I14A all increase the activation barrier for the chemical step by approximately 2 kcal/mol. The lack of an effect for S49A suggests that water structure is not important for stabilizing the hydride transfer transition state. In addition, the nominal effects observed for these mutations disfavor the hypothesis that neighboring amino acid residues participate in the stabilization of the reaction transition state through polar or weakly polar contacts.