title: Roles of quaternary structure and cysteine residues in the activity of human serine racemase.
abstract: BACKGROUND
  D-serine is an important coagonist at the NR1 subunit of the NMDA receptor class of glutamate receptors. It is chiefly synthesized in the CNS by serine racemase (SR). Regulation of SR activity is still poorly understood. As step toward developing reagents and methods for investigating SR in vitro, we analyzed structure-function relationships of a recombinant enzyme of human sequence.
  
  
  RESULTS
  Michaelis-Menten kinetic analysis indicated a KM value of 14 mM and Vmax value of 3.66 μmol·mg⁻¹·hr⁻¹ when L-serine was used as a substrate for purified SR. Gel-filtration chromatography and protein cross-linking experiments revealed that dimer is the major oligomeric form of recombinant SR in aqueous solution, though the proportions of monomer, tetramer, and larger aggregates differed somewhat with the specific buffer used. These buffers also altered activity in a manner correlating with the relative abundance of dimer. Activity assays showed that the dimeric gel-filtration fraction held the highest activity. Chemical reduction with DTT increased the activity of SR by elevating Vmax; cystamine, a reagent that blocks sulfhydryl groups, abolished SR activity. Gel-filtration chromatography and western blot analysis indicated that DTT enhanced the recovery of noncovalent SR dimer.
  
  
  CONCLUSIONS
  These data suggest that SR is most active as a noncovalent dimer containing one or more free sulfhydryls in the enzyme's active center or a modulatory site. Buffer composition and reduction/oxidation status during preparation can dramatically impact interpretations of SR activity. These findings also highlight the possibility that SR is sensitive to oxidative stress in vivo.
authors.affiliation: Department of Geriatrics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Steven W
authors.initials: SW
authors.lastname: Barger
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology*; D002478:Cells, Cultured; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D009457:Neuroglia / Q000201:enzymology; D010084:Oxidation-Reduction; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D019998:Racemases and Epimerases / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D000099300:Serine Racemase
chemical_list: D003545:Cysteine; D019998:Racemases and Epimerases; D011994:Recombinant Proteins; D000099300:Serine Racemase