Dual substrate and reaction specificity in mouse serine racemase: identification of high-affinity dicarboxylate substrate and inhibitors and analysis of the beta-eliminase activity.
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 racemization including glycine (K(I) = 1.63 mM), several dicarboxylic acids including malonate (K(I) = 0.077 mM), and l-erythro-3-hydroxyaspartate (K(I) = 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 beta-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 racemization 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 alpha-carboxyl and leaving groups. The possible implications of our observations for inhibitor design, regulation of activity, and function of mSR are discussed.
Strísovský
Kvido
K
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, Praha 6, 166 10, Czech Republic.
Jirásková
Jana
J
Mikulová
Adriana
A
Rulísek
Lubomír
L
Konvalinka
Jan
J
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular; D003998:Dicarboxylic Acids / Q000494:pharmacology*; D004791:Enzyme Inhibitors; D007700:Kinetics; D008190:Lyases / Q000378:metabolism; D051379:Mice; D019998:Racemases and Epimerases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity; D000099300:Serine Racemase
D000596:Amino Acids; D003998:Dicarboxylic Acids; D004791:Enzyme Inhibitors; D008190:Lyases; D019998:Racemases and Epimerases; D012694:Serine; D000099300:Serine Racemase