Hill M A, Kaufmann K, Otero J, Preiss J
Department of Biochemistry, Michigan State University, East Lansing 48824.
J Biol Chem. 1991 Jul 5;266(19):12455-60.
Site-directed mutagenesis was used to explore the role of Lys-195 in ADP-glucose pyrophosphorylase from Escherichia coli. This residue, which is conserved in every bacterial and plant source sequenced to date, was originally identified as a potential catalytic site residue by covalent modification studies. Mutation of Lys-195 to glutamine produces an enzyme whose Km for glucose 1-phosphate is 600-fold greater than that measured for the wild-type enzyme. The effect on glucose 1-phosphate is very specific since kinetic constants measured for ATP, Mg2+, and the allosteric activator, fructose 1,6-bisphosphate, are unchanged relative to those measured for the wild-type enzyme. Furthermore, the catalytic rate constant, Kcat, for the glutamine mutant is similar to that of the wild-type enzyme. Taken together, the results suggest a role for Lys-195 in binding of glucose 1-phosphate and exclude its role as a participant in the rate-determining step(s) in the catalytic reaction mechanism. To further study the effect of charge, shape, size, and hydrophobicity of the amino acid residue at position 195, a series of mutants were prepared including arginine, histidine, isoleucine, and glutamic acid. In every case, the kinetic constants measured for ATP, Mg2+, and fructose 1,6-bisphosphate were similar to wild-type constants, reinforcing the notion that this residue is responsible for a highly localized effect at the glucose 1-phosphate-binding site and also suggesting that the protein can accommodate a wide range of substitutions at this position without losing its global folding properties. Thermal stability measurements corroborate this finding. The mutations did, however, produce a range of glucose 1-phosphate Km values from 100- to 10,000-fold greater than wild-type, which indicate that both size and charge properties of lysine are essential for proper binding of glucose 1-phosphate at the catalytic site. AMP binding was also affected by the nature of the mutation at position 195. A model for glucose 1-phosphate, ATP, and AMP binding is presented.
定点诱变被用于探究大肠杆菌中赖氨酸 -195 在 ADP - 葡萄糖焦磷酸化酶中的作用。该残基在迄今测序的每一种细菌和植物来源中都是保守的，最初通过共价修饰研究被鉴定为潜在的催化位点残基。将赖氨酸 -195 突变为谷氨酰胺产生一种酶，其对磷酸葡萄糖的 Km 值比野生型酶测得的值大 600 倍。对磷酸葡萄糖的影响非常特异，因为相对于野生型酶测得的动力学常数，对 ATP、Mg2 + 和变构激活剂 1,6 - 二磷酸果糖测得的动力学常数没有变化。此外，谷氨酰胺突变体的催化速率常数 Kcat 与野生型酶相似。综合来看，结果表明赖氨酸 -195 在磷酸葡萄糖的结合中起作用，并排除了其作为催化反应机制中速率决定步骤参与者的作用。为了进一步研究 195 位氨基酸残基的电荷、形状、大小和疏水性的影响，制备了一系列突变体，包括精氨酸、组氨酸、异亮氨酸和谷氨酸。在每种情况下，对 ATP、Mg2 + 和 1,6 - 二磷酸果糖测得的动力学常数与野生型常数相似，强化了这一残基在磷酸葡萄糖结合位点负责高度局部效应的观点，也表明该蛋白质在这个位置可以容纳多种替代而不丧失其整体折叠特性。热稳定性测量证实了这一发现。然而，这些突变产生了一系列磷酸葡萄糖 Km 值，比野生型大 100 至 10000 倍，这表明赖氨酸的大小和电荷性质对于磷酸葡萄糖在催化位点的正确结合都是必不可少的。AMP 的结合也受到 195 位突变性质的影响。提出了一个磷酸葡萄糖、ATP 和 AMP 结合的模型。
