Multiple isotope effect probes of glutamate decarboxylase.
The enzymatic decarboxylation of glutamic acid shows a carbon isotope effect k12/k13 = 1.018 at 37 degree C, pH 4.7. In D2O under otherwise identical conditions, k12/k13 = 1.009. Under the same conditions solvent isotope effects are Vmax H2O/Vmax D2O = 5.0 and (Vmax/Km)H2O/(Vmax/Km)D2O = 2.6. With the assumption that the carbon isotope effect on the decarboxylation step is in the usual range (1.05--1.07), it is possible to derive relative rates and solvent isotope effects for all steps in the enzyme mechanism. Substrate binding in approximately 2-fold weaker in H2O than in D2O, probably because of the desolvation which accompanies binding of the substrate to the enzyme. A proton inventory analysis of the reaction shows that the Schiff base interchange has a large solvent isotope effect composed of relatively small contributions from at least four separate sites. A conformation change probably accompanies this step. The decarboxylation step shows a solvent isotope effect of approximately 2. Schiff base interchange and decarboxylation are both partially rate determining. The pH dependence of the isotope effects indicates that the initial step in the reaction can occur by way of two different pathways.
D002247:Carbon Isotopes; D002262:Carboxy-Lyases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005968:Glutamate Decarboxylase / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics
D002247:Carbon Isotopes; D005971:Glutamates; D002262:Carboxy-Lyases; D005968:Glutamate Decarboxylase