Understanding catalytic specificity in alanine racemase from quantum mechanical and molecular mechanical simulations of the arginine 219 mutant

Alanine racemase (AlaR) catalyzes the interconversion between l-Ala and d-Ala with the aid of the cofactor pyridoxal 5′-phosphate (PLP). The pyridine nitrogen in PLP in the wild-type enzyme is unprotonated due to interaction with Arg219, a rare feature among PLP-dependent enzymes. Herein, we performed combined quantum mechanics and molecular mechanics molecular dynamics simulations to study the Arg219Glu mutant AlaR. In this form of the enzyme, the PLP-pyridine nitrogen is protonated. This study suggests that the catalytic effect in the Arg219Glu mutant enzyme is due to a combined solvent and inherent stabilizing effect of the protonated cofactor, in contrast to the wild-type enzyme where the catalytic effect may be ascribed to solvent effects alone. Furthermore, we find that the quinonoid intermediate is greatly stabilized in the mutant enzyme, opening the possibility for side reactions such as transamination. We show that a computed 1,3-proton transfer in PLP due to the catalytic Lys39 is a feasible side reaction en route to transamination.