Abell L M, O'Leary M H
Department of Chemistry, University of Wisconsin-Madison 53706.
Biochemistry. 1988 May 3;27(9):3325-30. doi: 10.1021/bi00409a031.
The nitrogen isotope effect on the decarboxylation of glutamic acid by glutamate decarboxylase from Escherichia coli has been measured by comparison of the isotopic composition of the amino nitrogen of the product gamma-aminobutyric acid isolated after 10-20% reaction with that of the starting glutamic acid. At pH 4.7, 37 degrees C, the isotope effect is k14/k15 = 0.9855 +/- 0.0006 when compared to unprotonated glutamic acid. Interpretation of this result requires knowledge of the equilibrium nitrogen isotope effect for Schiff base formation. This equilibrium isotope effect is k14/k15 = 0.9824 for the formation of the unprotonated Schiff base between unprotonated valine and salicylaldehyde. Analysis of the nitrogen isotope effect on decarboxylation of glutamic acid and of the previously measured carbon isotope effect on this same reaction [O'Leary, M.H., Yamada, H., & Yapp, C.J. (1981) Biochemistry 20, 1476] shows that decarboxylation and Schiff base formation are jointly rate limiting. The enzyme-bound Schiff base between glutamate and pyridoxal 5'-phosphate partitions approximately 2:1 between decarboxylation and return to the starting state. The nitrogen isotope effect also reveals that the Schiff base nitrogen is protonated in this intermediate.
通过比较在10%-20%反应后分离得到的产物γ-氨基丁酸的氨基氮的同位素组成与起始谷氨酸的同位素组成，测定了氮同位素对大肠杆菌谷氨酸脱羧酶催化谷氨酸脱羧反应的影响。在pH 4.7、37℃条件下，与未质子化的谷氨酸相比，同位素效应为k14/k15 = 0.9855±0.0006。对该结果的解释需要了解席夫碱形成的平衡氮同位素效应。对于未质子化的缬氨酸与水杨醛之间形成未质子化席夫碱的反应，该平衡同位素效应为k14/k15 = 0.9824。对谷氨酸脱羧反应的氮同位素效应以及之前测定的该反应的碳同位素效应[O'Leary, M.H., Yamada, H., & Yapp, C.J. (1981) Biochemistry 20, 1476]的分析表明，脱羧反应和席夫碱形成共同构成速率限制步骤。谷氨酸与磷酸吡哆醛之间的酶结合席夫碱在脱羧反应和回到起始状态之间的分配比例约为2:1。氮同位素效应还表明，该中间体中的席夫碱氮是质子化的。
