Combined quantum mechanical/molecular mechanical simulations have been carried out to investigate the origin of the carbon acidity enhancement in the alanine racemization reaction catalyzed by alanine racemase (AlaR). The present study shows that the enhancement of carbon acidity of alpha-amino acids by the cofactor pyridoxal 5'-phosphate (PLP) with an unusual, unprotonated pyridine is mainly due to solvation effects, in contrast to the intrinsic electron-withdrawing stabilization by the pyridinium ion to form a quinonoid intermediate. Alanine racemase further lowers the R-proton acidity and provides an overall 14-17 kcal/mol transition-state stabilization. The second key finding of this study is that the mechanism of racemization of an alanine zwitterion in water is altered from an essentially concerted process to a stepwise reaction by formation of an external aldimine adduct with the PLP cofactor. Finally, we have used a centroid path integral method to determine the intrinsic kinetic isotope effects for the two proton abstraction reactions, which are somewhat greater than the experimental estimates.