The catalytic mechanism of a pyridoxal 5'-phosphate-dependent enzyme, L-serine dehydratase, has been investigated using ab initio quantum mechanical/molecular mechanical (QM/MM) methods. New insights into the chemical steps have been obtained, including the chemical role of the substrate carboxyl group in the Schiff base formation step and a proton-relaying mechanism involving the phosphate of the cofactor in the beta-hydroxyl-leaving step. The latter step is of no barrier and follows sequentially after the elimination of the a-proton, leading to a single but sequential a, beta-elimination step. The rate-limiting transition state is specifically stabilized by the enzyme environment. At this transition state, charges are localized on the substrate carboxyl group, as well as on the amino group of Lys4l. Specific interactions of the enzyme environment with these groups are able to lower the activation barrier significantly. One major difficulty associated with studies of complicated enzymatic reactions using ab initio QM/MM models is the appropriate choices of reaction coordinates. In this study, we have made use of efficient semiempirical models and pathway optimization techniques to overcome this difficulty.