Nitrogen isotope effects on glutamate decarboxylase from Escherichia coli

• Published in: Biochemistry (Easton) 1988-05, Vol.27 (9), p.3325-3330
• Main Authors: Abell, Lynn M, O'Leary, Marion H
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; bases; Biological and medical sciences; decarboxylation; Enzymes and enzyme inhibitors; Escherichia coli; Escherichia coli - enzymology; Fundamental and applied biological sciences. Psychology; glutamate decarboxylase; Glutamate Decarboxylase - metabolism; Isotope Labeling - methods; Kinetics; Lyases; Mathematics; nitrogen; Nitrogen Isotopes
• ISSN: 0006-2960; 1520-4995
• 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.