Mechanism of the reaction catalyzed by glutamate racemase.
The mechanism of the cofactor-independent glutamate racemase from Lactobacillus has been studied. The possible formation of an acylenzyme intermediate during catalysis has been investigated using 18O-carboxyl labeled glutamate. The absence of any washout of label during racemization argues against intermediate formation. The observation of the enzyme-catalyzed incorporation of deuterium at the C-2 position of glutamate upon racemization in D2O provides evidence for a deprotonation/protonation mechanism. Further experiments have been performed in order to determine the number of enzymic bases responsible for racemization. Solvent deuterium is efficiently incorporated into the product enantiomer but not into the recovered substrate enantiomer in each reaction direction. This finding is consistent with a "two-base" mechanism in which one enzymic base deprotonates the substrate, and the conjugate acid of a second enzymic base protonates the resulting intermediate from the opposite face. It also suggests that the two bases are monoprotic. The possibility that the two enzymic forms, which differ at the very least by the protonation states of the active-site bases, are kinetically significant has been examined by measuring the entire time course of the approach to equilibrium at various concentrations of glutamate. An "oversaturated" regime [Fisher, L. M., Albery, W. J., & Knowles, J. R. (1986) Biochemistry 25, 2529-2537] was not observed using glutamate concentrations as high as 100 mM, indicating that the two enzyme forms are rapidly interconverting under physiological conditions.
Gallo
K A
KA
Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138.
Tanner
M E
ME
Knowles
J R
JR
D000591:Amino Acid Isomerases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003903:Deuterium; D017666:Deuterium Oxide; D004926:Escherichia coli / Q000235:genetics; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D006859:Hydrogen; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010103:Oxygen Isotopes; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014867:Water
D005971:Glutamates; D010103:Oxygen Isotopes; D011994:Recombinant Proteins; D014867:Water; D018698:Glutamic Acid; D006859:Hydrogen; D003903:Deuterium; D000591:Amino Acid Isomerases; C062292:glutamate racemase; D017666:Deuterium Oxide