Thermodynamic and kinetic aspects of the reaction of gamma-fluoroglutamate with D-glutamate cyclase.

Diastereomers of y-fluoroglutamate tentatively assigned the D configuration at the or-carbon are substrates for Dglutamate cyclase purified from mouse liver. In order to elucidate properties of these potentially useful analogues of glutamate as well as to gain some insight into the mechanism of the enzyme, the kinetics and thermodynamics of the reaction of these analogues have been compared to those of glutamate. The Michaelis constant for the reaction with cis-3-fluoro-2-pyrrolidone-5-carboxylate (0.2 M) is comparable to that with 2-pyrrolidone-5-carboxylate (0.09 M) but that of trans-3-fluoro-2-pyrrolidone&carboxylate (1.5 M) is distinctly higher. The equilibrium favors pyrrolidone formation for 2-pyrrolidone-5-carboxylate (97 %) and for trans-3-fluoro-2-pyrrolidoneJ-carboxylate (60%) but not for cis-3-fluoro-2-pyrrolidone-5-carboxylate (30%). The lactamization of glutamate has an enthalpy change of +2.3 kcal per mole while that for the diastereomer of y-fluoroglutamate forming the cis pyrrolidone is +3.8 kcal per mole. The fluorine substituent greatly increases the V,,,,, for the enzymatic hydrolysis in both diastereomers of the pyrrolidone. The relevance of these observations to the mechanism of the enzyme and the conformation of the substrates is discussed.