Mechanism of eukaryotic serine racemase-catalyzed serine dehydration.

Eukaryotic serine racemase (SR) is a pyridoxal 5'-phosphate enzyme belonging to the Fold-type II group, which catalyzes serine racemization and is responsible for the synthesis of D-Ser, a co-agonist of the N-methyl-d-aspartate receptor. In addition to racemization, SR catalyzes the dehydration of D- and L-Ser to pyruvate and ammonia. The bifuctionality of SR is thought to be important for D-Ser homeostasis. SR catalyzes the racemization of D- and L-Ser with almost the same efficiency. In contrast, the rate of L-Ser dehydration catalyzed by SR is much higher than that of D-Ser dehydration. This has caused the argument that SR does not catalyze the direct D-Ser dehydration and that D-Ser is first converted to L-Ser, then dehydrated. In this study, we investigated the substrate and solvent isotope effect of dehydration of D- and L-Ser catalyzed by SR from Dictyostelium discoideum (DdSR) and demonstrated that the enzyme catalyzes direct D-Ser dehydration. Kinetic studies of dehydration of four Thr isomers catalyzed by D. discoideum and mouse SRs suggest that SR discriminates the substrate configuration at C3 but not at C2. This is probably the reason for the difference in efficiency between L- and D-Ser dehydration catalyzed by SR.

Reaction mechanism of Zn2+-dependent d-serine dehydratase: role of a conserved tyrosine residue interacting with pyridine ring nitrogen of pyridoxal 5'-phosphate.

d-Serine dehydratase from Saccharomyces cerevisiae (Dsd1p) is a pyridoxal 5'-phosphate (PLP)- and Zn(2+)-dependent enzyme that catalyzes the dehydration of d-serine to yield pyruvate and ammonia. Dsd1p uses the Tyr residue (Y203) to interact with the pyridine nitrogen of PLP, which is a unique feature of PLP enzymes. To investigate the role of Y203 in catalysis, a series of Y203 mutants was constructed and studied. Mutant enzymes possessing a non-polar or a basic residue instead of Y203 (Y203F, A, S and R) exhibited substantial levels of catalytic activity, and among these, the Y203F mutant had the least impact on catalytic activity. The Y203D exhibited a 10(5)-fold decrease in enzyme activity, and unlike wild-type enzyme, the mutant enzyme favoured the Cα reprotonation before hydroxyl group protonation. Our data show that the Y203 does not participate in the protonation of the pyridine nitrogen (N1) of PLP, and Dsd1p uses the cofactor in an N1-unprotonated state. The unprotonated N1 promotes elimination of the leaving group and evades Cα reprotonation before hydroxyl group protonation.