Karsten W E, Chooback L, Cook P F
Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019, USA.
Biochemistry. 1998 Nov 10;37(45):15691-7. doi: 10.1021/bi9812827.
Site-directed mutagenesis was used to change E190 of sheep liver 6-phosphogluconate dehydrogenase to A, D, H, K, Q, and R to probe its possible role as a general acid catalyst. Each of the mutant proteins was characterized with respect to the pH dependence of kinetic parameters. Mutations that eliminate a titrable group at position 190, result in pH-rate profiles with no observable pK on the basic side of the V/K6PG profile. Mutations that change the pK of the group at position 190 result in the expected pK perturbations in the V/K6PG profile. Kinetic parameters obtained at the pH optimum in the pH-rate profiles are consistent with a rate-limiting tautomerization of the 1,2-enediol of ribulose 5-phosphate consistent with the proposed role of E190. Data are also consistent with some participation of E190 in an isomerization required to form the active Michaelis complex.
采用定点诱变技术将绵羊肝脏6-磷酸葡萄糖酸脱氢酶的E190分别替换为A、D、H、K、Q和R，以探究其作为广义酸催化剂的可能作用。对每个突变蛋白的动力学参数的pH依赖性进行了表征。消除190位可滴定基团的突变会导致pH速率曲线在V/K6PG曲线的碱性一侧没有可观察到的pK。改变190位基团pK的突变会导致V/K6PG曲线中预期的pK扰动。在pH速率曲线的最佳pH值下获得的动力学参数与5-磷酸核酮糖1,2-烯二醇的限速互变异构一致，这与E190的假定作用相符。数据还表明E190在形成活性米氏复合物所需的异构化过程中也有一定参与。
