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.

Figure 1

Kinetic characterization of recombinant human SR. A. Michaelis-Menten plot of the activity of recombinant SR versus increasing concentrations of L-Ser; B. Double-reciprocal plot (Lineweaver-Burk) of the data with the axes 1/V versus 1/[L-Ser]. SR activity assay was performed as described under "Methods".

Figure 2

Gel-filtration chromatography of recombinant SR. Purified recombinant SR with affinity column was extensively dialyzed against 50 mM phosphate buffer, pH 8.0, 52 mM NaCl. After concentration with Centricon filters, aliquots of 50 μl of ~3 mg/ml of recombinant SR were loaded onto a Superdex HR200 gel-filtration column. Separation was performed at 25°C with flow rate at 0.5 ml/min. The elution buffer was the same as dialysis buffer. Protein detection was performed at 280 nm.

Figure 3

Stabilization of in vitro- and in vivo-formed multimers by chemical cross-linker. A. Increasing concentrations of BS³ were incubated with 12.5 μM recombinant SR on ice for 2 h, then the reactions were quenched by addition of Tris-HCl. Reaction samples were analyzed on 10% SDS-PAGE with 2.5% β-mercaptoethanol in the sample buffer. B. Increasing concentrations of protein cross-linker BS³ were incubated with lysates from mouse primary astrocytes (0.5 μg protein/μl) on ice for 2 h, and the reactions were quenched by the addition of Tris-HCl. The formation of cross-linked SR protein was analyzed by western blot with an anti-SR antibody.

Figure 4

Influence of buffer components on SR activity. Recombinant SR purified by affinity chromatography was dialyzed against a phosphate-buffered (solid line) or Tris-buffered solution (dashed line). Then, SR activity of each enzyme preparation was monitored as described under "Methods" except for the reaction buffers, which corresponded to each respective dialysis buffer.

Figure 5

Influence of buffer components on quaternary structure of recombinant SR. Recombinant SR purified by affinity chromatography was dialyzed against a phosphate-buffered (solid line) or Tris-buffered solution (dashed line). After concentration with Centricon filters, 50-μl aliquots of ~3 mg/ml of each recombinant SR were loaded onto a Superdex HR200 gel-filtration column and eluted with same buffer as the corresponding dialysis buffer.

Figure 6

Chemical reduction elevates SR activity by increasing its V_max. SR activity assay was performed in the phosphate buffer with 2.5 μg recombinant enzyme and the indicated concentrations of L-serine; DTT was alternatively excluded (solid line) or included (dashed line) at 0.2 mM. After incubation at 37°C for 30 min, the reaction was terminated by boiling for 5 min. D-ser formation was monitored by a chemiluminescence assay. The data are representative of three different protein preparations.

Figure 7

Effect of chemical reduction on quaternary structure of recombinant SR: native conditions. Recombinant SR purified by affinity chromatography was dialyzed against the phosphate buffer with (dashed line) or without (solid line) 0.2 mM DTT. After concentration with Centricon filters, 50-μl aliquots of ~10 mg/ml of each recombinant SR were injected onto a Superdex 200 column and eluted with the same buffer as the dialysis buffer.

Figure 8

Effect of chemical reduction on quaternary structure of recombinant SR: denaturing conditions. A. Recombinant SR was dialyzed against phosphate buffers with or without 0.2 mM DTT after elution from the nickel affinity column. B. SR preparations in panel A were injected onto a Superdex HR200 column and eluted with same buffer as the corresponding dialysis buffer. Recombinant SR oligomeric species of both protein preparations in A and B were subjected to SDS-PAGE in presence or absence of 0.2 mM DTT in the sample loading buffer. The gels were then immunoblotted with anti-SR antibody.

Figure 9

The role of free sulfhydryls in activity of SR dimers. Dimeric fractions of recombinant SR were eluted from a Superdex 200 column with or without DTT (0.2 mM) in the mobile phase. SR activity was then assayed. The -DTT fraction was assayed with or without DTT (0.2 mM) or cystamine (1 mM) in the reaction buffer. (**p < 0.0001).

Figure 10

SR protein from a cellular source oxidizes to dimers and other multimers. Total cellular protein was prepared from three cultures of mouse primary astrocytes and resolved by 10% SDS-PAGE in the absence (A) or presence (B) of 2.5% β-mercaptoethanol in the sample buffer. SR protein (arrow) was detected by western blot with anti-SR antibody. Lane 1 contained molecular weight standards (kDa).