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J. Biol. Chem., Vol. 277, Issue 31, 27782-27792, August 2, 2002
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From the Departamento de Bioquímica y Biología
Molecular, Fac. CC. Químicas, Universidad Complutense de
Madrid, Madrid 28040, Spain
Serine racemase (SR) is a brain enzyme present in
glial cells, where it isomerizes L-serine into
D-serine that, in turn, diffuses and coactivates the
N-methyl-D-aspartate receptor through
the binding to the so-called "glycine site." We have developed a
method for the slow expression of SR in a eukaryotic vector that
permits the correct insertion of the prosthetic group into the active site, rendering functional SR with a Km toward
L-serine of 4.8 mM. Divalent cations such as
calcium or manganese were necessary for complete enzyme activity,
whereas the presence of chelators such as EDTA completely inhibited the
enzyme. Moreover, direct binding of calcium to SR was evidenced using
45Ca2+. Gel filtration of the recombinant SR
revealed the protein to be in a dimer-tetramer equilibrium. The
addition of EDTA to a calcium-saturated serine racemase evokes a
profound conformational change, as monitored by both fluorescence and
circular dichroism techniques. Fluorescence titration allowed us to
calculate a binding constant for calcium of 6.2 µM.
Reagents that react with sulfhydryl groups, such as cystamine, were
potent inhibitors of SR, in a clear reflection that one or more
cysteine residues are important for enzyme activity. Additionally, 16 serine analogues were tested as a putative SR substrate or inhibitors.
Significant inhibition was only observed for
L-Ser-O-sulfate, L-cycloserine, and
L-cysteine. Finally, activation of brain SR as a result of
the changes in calcium concentration was studied in primary astrocytes.
Treatment of astrocytes with the calcium ionophore A23187, as well as with compounds that augment the intracellular calcium levels such as
glutamate or kainate led to an increase in the amount of
D-serine present in the extracellular medium. These results
suggest that there might be a glutamatergic-mediated regulation of SR
activity by intracellular calcium concentration.
Direct Calcium Binding Results in Activation of Brain
Serine Racemase*
,
*
This work was supported by Comunidad Autónoma de
Madrid Grants 08.4/0039.1/2000 and Ministerio de Ciencia y
Tecnología Grant BMC2000-055.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present address: Dept. of Chemistry, Columbia University, New
York, NY 10027.
§
To whom correspondence should be addressed. Tel.: 34-91-394-4258;
Fax: 34-91-394-4159; E-mail: nacho@bbm1.ucm.es.
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