Rosenberg R M, O'Leary M H
Biochemistry. 1985 Mar 26;24(7):1598-603. doi: 10.1021/bi00328a004.
We have measured the 13C kinetic isotope effect at pH 4.0, 5.0, 6.0, and 6.5 and in D2O at pD 5.0 and the rate of D-H exchange of the alpha and beta protons of aspartic acid in D2O at pD 5.0 for the reaction catalyzed by the enzyme aspartate beta-decarboxylase from Alcaligenes faecalis. The 13C kinetic isotope effect, with a value of 1.0099 +/- 0.0002 at pH 5.0, is less than the intrinsic isotope effect for the decarboxylation step, indicating that the decarboxylation step is not entirely rate limiting. We have been able to estimate probable values of the relative free energies of the transition states of the enzymatic reaction up to and including the decarboxylation step from the 13C kinetic isotope effect and the rate of D-H exchange of alpha-H. The pH dependence of the kinetic isotope effect reflects the pKa of the pyridine nitrogen of the coenzyme pyridoxal 5'-phosphate but not that of the imine nitrogen. A mechanism is proposed for the exchange of aspartate beta-H that is consistent with the stereochemistry suggested earlier.
我们测定了粪产碱杆菌天冬氨酸β-脱羧酶催化反应在pH 4.0、5.0、6.0和6.5条件下以及在重水（pD 5.0）中的13C动力学同位素效应，还测定了在重水（pD 5.0）中天冬氨酸α和β质子的D-H交换速率。在pH 5.0时，13C动力学同位素效应的值为1.0099±0.0002，小于脱羧步骤的内在同位素效应，这表明脱羧步骤并非完全是限速步骤。我们能够根据13C动力学同位素效应和α-H的D-H交换速率估算出直至并包括脱羧步骤的酶促反应过渡态相对自由能的可能值。动力学同位素效应的pH依赖性反映了辅酶磷酸吡哆醛吡啶氮的pKa，而不是亚胺氮的pKa。提出了一种与先前提出的立体化学一致的天冬氨酸β-H交换机制。
