Shim J H, Benkovic S J
Department of Chemistry, The Pennsylvania State University, University Park 16802, USA.
Biochemistry. 1999 Aug 3;38(31):10024-31. doi: 10.1021/bi9904609.
Site-directed mutagenesis followed by studies of the pH dependence of the kinetic parameters of the mutants has been used to probe the role of the active site residues and loops in catalysis by glycinamide ribonucleotide transformylase (EC 2.1.2.2). The analysis of the mutants of the strictly conserved active site residues, His108 and Asp144, revealed that His108 acts in a salt bridge with Asp144 as a general acid catalyst with a pK(a) value of 9.7. Asp144 also plays a key role in the preparation of the active site geometry for catalysis. The rate-limiting step in the pH range of 6-10 appears to be the catalytic steps involving tetrahedral intermediates, supported by the observation of a pL (L being H or D)-independent solvent deuterium isotope effect of 2. The ionization of the amino group of glycinamide ribonucleotide both as a free and as a bound form dominates the kinetic behavior at low pH. The analysis of a mutation, H121Q, within the loop spanning amino acids 111-131 suggests the closure of the loop is involved in the binding of the substrate. The kinetic behavior parallels pH effects revealed by a series of X-ray crystallographic structures of the apoenzyme and inhibitor-bound enzyme [Su, Y., Yamashita, M. M., Greasley, S. E. , Mullen, C. A., Shim, J. H., Jennings, P. A., Benkovic, S. J., and Wilson, I. A. (1998) J. Mol. Biol. 281, 485-499], permitting a more exact formulation of the probable catalytic mechanism.
通过定点诱变，随后研究突变体动力学参数的pH依赖性，已被用于探究甘氨酰胺核糖核苷酸转甲酰基酶（EC 2.1.2.2）活性位点残基和环在催化过程中的作用。对严格保守的活性位点残基His108和Asp144的突变体分析表明，His108与Asp144形成盐桥，作为pK(a)值为9.7的一般酸催化剂。Asp144在为催化作用准备活性位点几何结构方面也起着关键作用。在6 - 10的pH范围内，限速步骤似乎是涉及四面体中间体的催化步骤，这由观察到的2的pL（L为H或D）无关的溶剂氘同位素效应所支持。甘氨酰胺核糖核苷酸氨基的游离形式和结合形式的电离在低pH时主导动力学行为。对跨越氨基酸111 - 131的环内的H121Q突变的分析表明，环的闭合参与底物的结合。动力学行为与一系列脱辅基酶和抑制剂结合酶的X射线晶体结构所揭示的pH效应相似[Su, Y., Yamashita, M. M., Greasley, S. E., Mullen, C. A., Shim, J. H., Jennings, P. A., Benkovic, S. J., and Wilson, I. A. (1998) J. Mol. Biol. 281, 485 - 499]，从而能够更精确地阐述可能的催化机制。
