Human Serine Racemase Structure/Activity Relationship Studies Provide Mechanistic Insight and Point to Position-84 as a Hotspot for {beta}-Elimination Function [Protein Structure and Folding]

There is currently great interest in human serine racemase (hSR), the enzyme responsible for producing the NMDA co-agonist D-serine. Reported correlation of D-serine levels with disorders including Alzheimers disease, ALS and ischemic brain damage (elevated D-serine), and schizophrenia (reduced D-serine) has further piqued this interest. Reported is a structure/activity relationships (SAR) study of position-(S)84, 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 (L-SOS; ~1200-fold change in kcat/Km ratios) and L-threo-β-hydroxyaspartate (L-THA; ~5000-fold change in kcat/Km ratios) alternative substrates. On the other hand, the S84T (performs L-Ser-racemization activity), S84A (good kcat by 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 to the native enzyme. Inhibition studies with L-EHA follow this trend with both wt-SR and the SD4N 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 R135 in binding and properly positioning the L-THA and L-SOS substrates and the L-EHA inhibitor. Examination of available sequence data suggests that R135 may have originated for L-THA-like β-elimination function in earlier evolutionary variants. And examination of available structural data suggests that an S84-H2O-K114 H-bonding network in hSR lowers the pKa of the S84 re-face base.

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