Roles of quaternary structure and cysteine residues in the activity of human serine racemase

Wei Wang,Steven W Barger

doi:10.1186/1471-2091-12-63

Abstract

BackgroundD-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.ResultsMichaelis-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.ConclusionsThese 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.

Highlights

D-serine is an important coagonist at the NR1 subunit of the NMDA receptor class of glutamate receptors
serine racemase (SR) activity is augmented by physical interactions with other proteins, such as glutamate-receptor-interacting protein (GRIP) [22,23] and protein interacting with C kinase (PICK1) [24,25]
Purification and enzymatic characterization of recombinant human SR For these studies, SR cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from human RNA and cloned into pTrcHisB, a prokaryotic expression vector that encodes an in-frame polyhistidine domain at the protein’s N-terminus

Summary

Introduction

D-serine is an important coagonist at the NR1 subunit of the NMDA receptor class of glutamate receptors. 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 altered activity in a manner correlating with the relative abundance of dimer. When substantial levels of endogenous D-serine in the mammalian brain were discovered by Hashimoto et al [1], the source of this unusual amino acid became a critical question This intrigue was settled when Wolosker et al [2] cloned the full-length mouse serine racemase (SR) gene and purified the enzyme from rat brain. SR activity is augmented by physical interactions with other proteins, such as glutamate-receptor-interacting protein (GRIP) [22,23] and protein interacting with C kinase (PICK1) [24,25]

Methods

Results

Discussion

Conclusion