Search

Serine racemase (SR) is the first racemase enzyme to be identified in human biology and converts L-serine to D-serine, an important neuronal signaling molecule that serves as a co-agonist of the NMDA (N-methyl-D-aspartate) receptor. This overview describes key molecular features of the enzyme, focusing on the side chains and binding motifs that control PLP (pyridoxal phosphate) cofactor binding as well as activity modulation through the binding of both divalent cations and ATP, the latter showing allosteric modulation. Discussed are catalytically important residues in the active site including K56 and S84-the si- and re-face bases, respectively,-and R135, a residue that appears to play a critical role in the binding of both negatively charged alternative substrates and inhibitors. The interesting bifurcated mechanism followed by this enzyme whereby substrate L-serine can be channeled either into D-serine (racemization pathway) or into pyruvate (β-elimination pathway) is discussed extensively, as are studies that focus on a key loop region (the so-called "triple serine loop"), the modification of which can be used to invert the normal in vitro preference of this enzyme for the latter pathway over the former. The possible cross-talk between the PLP enzymes hSR and hCBS (human cystathionine β-synthase) is discussed, as the former produces D-serine and the latter produces H2S, both of which stimulate the NMDAR and both of which have been implicated in neuronal infarction pursuant to ischemic stroke. Efforts to gain a more complete mechanistic understanding of these PLP enzymes are expected to provide valuable insights for the development of specific small molecule modulators of these enzymes as tools to study their roles in neuronal signaling and in modulation of NMDAR function.

There is currently great interest in human serine racemase, the enzyme responsible for producing the NMDA co-agonist d-serine. Reported correlation of d-serine levels with disorders including Alzheimer's disease, ALS, and ischemic brain damage (elevated d-serine) and schizophrenia (reduced d-serine) has further piqued this interest. Reported here is a structure/activity relationship study of position Ser84, the putative re-face base. In the most extreme case of functional reprogramming, the S84D mutant displays a dramatic reversal of β-elimination substrate specificity in favor of l-serine over the normally preferred l-serine-O-sulfate (∼1200-fold change in kcat/Km ratios) and l (l-THA; ∼5000-fold change in kcat/Km ratios) alternative substrates. On the other hand, the S84T (which performs l-Ser racemization activity), S84A (good kcat but high Km for l-THA elimination), and S84N mutants (nearly WT efficiency for l-Ser elimination) displayed intermediate activity, all showing a preference for the anionic substrates, but generally attenuated compared with the native enzyme. Inhibition studies with l-erythro-β-hydroxyaspartate follow this trend, with both WT serine racemase and the S84N mutant being competitively inhibited, with Ki = 31 ± 1.5 μm and 1.5 ± 0.1 mm, respectively, and the S84D being inert to inhibition. Computational modeling pointed to a key role for residue Arg-135 in binding and properly positioning the l-THA and l-serine-O-sulfate substrates and the l-erythro-β-hydroxyaspartate inhibitor. Examination of available sequence data suggests that Arg-135 may have originated for l-THA-like β-elimination function in earlier evolutionary variants, and examination of available structural data suggests that a Ser84-H2O-Lys114 hydrogen-bonding network in human serine racemase lowers the pKa of the Ser84 re-face base.