The pyridoxal phosphate dependent alanine racemase catalyzes the interconversion of L- and D-alanine. The latter is an essential component of peptidoglycan in cell walls of Gram-negative and -positive bacteria, making alanine racemase an attractive target for antibacterials. Global analysis of protiated and deuterated progress curves simultaneously enables determination of intrinsic kinetic and equilibrium isotope effects for alanine racemase. The intrinsic primary kinetic isotope effects for Cα hydron abstraction are 1.57 ± 0.05 in the D → L direction and 1.66 ± 0.09 in the L → D direction. Secondary kinetic isotope effects were found for the external aldimine formation steps in both the L → D (1.13 ± 0.05, forward; 0.90 ± 0.03, reverse) and D → L (1.13 ± 0.06, forward; 0.89 ± 0.03, reverse) directions. The secondary equilibrium isotope effects calculated from these are 1.26 ± 0.07 and 1.27 ± 0.07 for the L -D and D → L directions, respectively. These equilibrium isotope effects imply substantial ground-state destabilization of the C-H bond via hyperconjugation with the conjugated Schiff base/pyridine ring π system. The magnitudes of the intrinsic primary kinetic isotope effects, the lower boundary on the energy of the quinonoid intermediate, and the protonation states of the active site catalytic acids/bases (K39-∈NH2 and Y265-OH) suggest that the pKa of the substrate Cα-H bond in the external aldimine lies between those of the two catalytic bases, such that the proton abstraction transition state is early in the D → L direction and late in the L → D direction.
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