Lee A Y, Zhang S, Kongsaeree P, Clardy J, Ganem B, Erickson J W, Xie D
Structural Biochemistry Program, SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702-1201, USA.
Biochemistry. 1998 Jun 23;37(25):9052-7. doi: 10.1021/bi980217u.
Electrostatic interactions play important roles in the catalysis of chorismate to prephenate by chorismate mutase. Mutation of Gln88 to glutamate in the monofunctional chorismate mutase from Escherichia coli results in an enzyme with a pH profile of activity significantly different from that of the wild type protein. To investigate whether the mutation alters the substrate binding process or the catalysis, we have directly determined the thermodynamic parameters of a transition state analogue inhibitor binding to the wild-type chorismate mutase and its Q88E mutant using isothermal titration calorimetry. The results demonstrate that solvent reorganization and hydrophobic interactions contribute the predominant free energy to inhibitor binding. The charge state of Glu88 in the Q88E mutant was experimentally determined and was shown to be protonated at pH 4.5 and ionized at pH 7.8, consistent with earlier hypotheses. Most surprisingly, inhibitor binding energetics do not exhibit significant pH dependency for both enzymes. Our findings indicate that the charge state of Glu88 has a small impact on inhibitor binding but plays an important role in the catalytic process.
静电相互作用在分支酸变位酶催化分支酸生成预苯酸的过程中发挥着重要作用。将来自大肠杆菌的单功能分支酸变位酶中的谷氨酰胺88突变为谷氨酸，会产生一种酶，其活性的pH曲线与野生型蛋白的显著不同。为了研究该突变是否改变底物结合过程或催化作用，我们使用等温滴定量热法直接测定了过渡态类似物抑制剂与野生型分支酸变位酶及其Q88E突变体结合的热力学参数。结果表明，溶剂重组和疏水相互作用为抑制剂结合贡献了主要的自由能。通过实验确定了Q88E突变体中谷氨酸88的电荷状态，结果显示其在pH 4.5时质子化，在pH 7.8时电离，这与早期的假设一致。最令人惊讶的是，两种酶的抑制剂结合能量学均未表现出显著的pH依赖性。我们的研究结果表明，谷氨酸88的电荷状态对抑制剂结合影响较小，但在催化过程中起重要作用。
