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.