Butler-Ransohoff J E, Kendall D A, Kaiser E T
Laboratory of Bioorganic Chemistry and Biochemistry, Rockefeller University, New York, NY 10021-6399.
Proc Natl Acad Sci U S A. 1988 Jun;85(12):4276-8. doi: 10.1073/pnas.85.12.4276.
The guanidinium group of arginine-166 has been postulated to act as an electrophilic species during phosphorylation of alkaline phosphatase. Its role could be either to stabilize the developing negative charge on the oxygen of the leaving group or the pentacoordinate transition state or to help bind the -PO2-3 group. We have produced via site-directed mutagenesis two Escherichia coli alkaline phosphatase mutants (lysine-166 and glutamine-166) to test whether the guanidinium group plays a critical role in catalysis. Comparative kinetic characterization of the lysine-166 and glutamine-166 mutants indicates that the charge at residue 166 is not required for the hydrolysis of phosphate monoesters. Small decreases in kcat are observed for both the lysine and glutamine mutants, relative to the wild-type enzyme, but the value for the uncharged glutamine mutant is only one-third that of lysine. Thus, the stabilizing effect of the positively charged guanidinium group does not appear to play a major role in the rate-limiting step for substrate hydrolysis. A significant effect on the Km value is seen only for the glutamine mutant.
精氨酸166的胍基被假定在碱性磷酸酶磷酸化过程中作为亲电物质起作用。它的作用可能是稳定离去基团氧上逐渐形成的负电荷或五配位过渡态，或者帮助结合 -PO2-3基团。我们通过定点诱变产生了两种大肠杆菌碱性磷酸酶突变体（赖氨酸-166和谷氨酰胺-166），以测试胍基在催化中是否起关键作用。赖氨酸-166和谷氨酰胺-166突变体的比较动力学特征表明，166位残基的电荷对于磷酸单酯的水解不是必需的。相对于野生型酶，赖氨酸和谷氨酰胺突变体的kcat均有小幅下降，但不带电荷的谷氨酰胺突变体的值仅为赖氨酸的三分之一。因此，带正电荷的胍基的稳定作用似乎在底物水解的限速步骤中不发挥主要作用。仅谷氨酰胺突变体对Km值有显著影响。
