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Abstract

Serine racemase, purified from mouse brain, consisted of two isoforms. They had similar enzymatic properties and had molecular weights of about 55 kDa based on size exclusion chromatography. This is about twice that reported from its electrophoretic mobility on SDS gels or from the amino acid sequence of the recombinant enzyme. In addition to the previously reported requirements for pyridoxal phosphate and reducing agents, we found that both forms of the enzyme required Mg2+ and were strongly stimulated by yeast extract. The yeast extract could be replaced by ATP, GTP, or ADP and, to a lesser extent, by other nucleotides. In the presence of 1 mM ATP, the Km for L-serine decreased from 13 mM to 1.8 mM with little change in Vmax, indicating an allosteric mechanism for nucleotide activation. In addition to acting as a serine racemase, the enzyme has been reported to act on L-serine O-sulfate as a dehydratase. When measured by HPLC, after derivatization with 2,4 dinitrophenylhydrazine, we found, as expected, a very rapid formation of pyruvate from this substrate. L-serine was also converted to pyruvate at about twice the racemization rate. L-serine O-sulfate dehydration was inhibited by ATP, while L-serine dehydration, like racemization, was activated by nucleotides, indicating that, for L-serine, dehydration and racemization take place at the same site.

Random mutagenesis of human serine racemase reveals residues important for the enzymatic activity

“…The oligomerization state of wild-type SR and the mutants was accessed by gel filtration on Superdex200HR as described in Experimental. The wild-type, K221E, and C217S mutants eluted as a single major peak corresponding to roughly 60 kDa, which is in accord with the results of Dunlop and Neidle 3 . While this is slightly lower than the predicted molecular weight of a SR dimer (74 kDa), it is significantly larger than the molecular weight of the monomer (37 kDa), and SR has been reported to form homodimers by several groups 1,3,5 .…”

“…In addition to its racemase activity, SR is also involved in serine metabolism by acting as a β-eliminase, converting L-serine [1][2][3] , and to a lesser extent D-serine 4 , to pyruvate, with concomitant ammonia release. Both the racemization and elimination activities of mammalian SR are upregulated by divalent cations 1,5 , ATP 3 , and reducing agents 6 .…”

A Novel Pyridoxal 5′-Phosphate-dependent Amino Acid Racemase in the Aplysia californica Central Nervous System

“…4C (34) and AMP promoted SbAspR activity, but ATP reduced it (35). It is interesting that while ATP has a positive effect on DAR1 activity, the same nucleotide showed an inhibitory effect on SbAspR activity.…”

“…The enzyme is PLP-dependent and appears to require dimerization for activity. Homodimers have been observed with mouse SerR and SbAspR (32,34,40). The recombinant DAR1 appears to be heat-tolerant under the assay conditions used.…”

“…When both effectors were present, the enzyme showed the largest activity for the D-Asp and D-Ser conversions. The ATP and MgCl 2 concentrations were not optimized for DAR1; we chose the concentrations based on published research on Ser and Asp racemases (34,35). Finally, Fig.…”