Dual substrate and reaction specificity in mouse serine racemase: Identification of high-affinity dicarboxylate substrate and inhibitors and analysis of the beta-eliminase activity.
Strisovsky, K.; Jiraskova, J.; Mikulova, A. etc.,
Biochemistry, 44  13091 - 13100 (2005)
Mouse serine racemase (mSR) is a pyridoxal 5'-phosphate dependent enzyme that catalyzes the biosynthesis of the N-methyl-D-aspartate receptor coagonist D-serine in the brain. Furthermore, mSR catalyzes beta-elimination of serine and L-serine-O-sulfate into pyruvate. The biological significance of this beta-elimination activity and the factors influencing mSR substrate and reaction specificity, which are crucial for prospective inhibitor design, are poorly understood. Using a bacterial expression system and ATP-agarose affinity chromatography, we have generated a pure and active recombinant mSR and investigated its substrate and reaction specificity in vitro by analyzing a systematic series of compounds derived from L-Ser and L-serine-O-sulfate. The analysis revealed several competitive inhibitors of serine racernization including glycine (K-I = 1.63 mM), several dicarboxylic acids including malonate (K-I = 0.077 mM), and L-erythro-3-hydroxyaspartate (KI = 0.049 mM). The latter compound represents the most effective inhibitor of SR reported to date. A simple inversion of the beta-carbon configuration of the compound yields an excellent P-elimination substrate L-threo-3-hydroxyaspartate. Inhibition analysis indicates that racemization and beta-elimination activities of mSR reside at the same active site. While the racernization activity is specific to serine, the beta-elimination activity has a broader specificity for L-amino acids with a suitable leaving group at the beta-carbon and optimal spatial orientation of the a-carboxyl and leaving groups. The possible implications of our observations for inhibitor design, regulation of activity, and function of mSR are discussed.