Direct calcium binding results in activation of brain serine racemase.
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 K(m) 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 (45)Ca(2+). 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 microm. 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, 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.
Cook
Silas P
SP
Departamento de Bioquímica y Biología Molecular, Fac. CC. Químicas, Universidad Complutense de Madrid, Madrid 28040, Spain.
Galve-Roperh
Ismael
I
Martínez del Pozo
Alvaro
A
Rodríguez-Crespo
Ignacio
I
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D002942:Circular Dichroism; D003523:Cycloserine / Q000494:pharmacology; D003545:Cysteine / Q000494:pharmacology; D017931:DNA Primers; D004789:Enzyme Activation; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D016254:Mutagenesis, Insertional; D011487:Protein Conformation; D019998:Racemases and Epimerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D000099300:Serine Racemase
D002118:Calcium; D002413:Cations, Divalent; D003523:Cycloserine; D003545:Cysteine; D017931:DNA Primers; D008565:Membrane Proteins; D019998:Racemases and Epimerases; D011994:Recombinant Proteins; D000099300:Serine Racemase