Human serine racemase, a key enzyme in neurophysiology and neuropathologies

D-Ser is the co-agonist of the NMDA receptor, mediating key neu-rological functions. D-Ser is synthesized by serine racemase (SR), apyridoxal 5′-phosphate (PLP)-dependent enzyme catalyzing boththe reversible racemisation of L-Ser to D-Ser and the beta-elimina-tion of both enantiomers in the mammalian brain1. SR activity isregulated by ATP, divalent cations, post-translational modifica-tions and interacting proteins. Because high and low D-Ser levelsare associated with distinct neuropathologies1, the elucidation ofthe molecular basis of SR catalysis and regulation is instrumentalto the design of effectors for tailored therapy1–3.We have carried out experiments on purified recombinant humanSR in the presence of 1 mM Mg++, and in the absence and pres-ence of ATP. In the beta-elimination ATP causes a nearly six-folddecrease in the KMfor L-Ser and a five-fold increase in kcat,yieldinga 30-fold increase in kcat/KM.The activity is increased almost two-fold by LiCl, NaCl, KCl, NH4Cl (at 150 mM), with respect to theabsence of monovalent ions and 3 mM chloride. This effect ismainly caused by the anion because it was found an activity increasewith decreasing halide size in the series F ,Cl ,Br and I .The absorbance band of SR is centered at 412 nm, indicatingthat the ketoenamine tautomer of PLP is the predominant spe-cies. Excitation of Trp leads to an intense fluorescence peak at341 nm and a weaker peak at 500 nm, also excited directly at412 nm. The intensity change at 500 nm as a function of ligandconcentration was exploited for the determination of the Kdissofglycine and malonate, two known SR inhibitors. The Kdissof gly-cine is 7.2 0.3 mM and 0.49 0.03 mM in the absence andpresence of ATP, respectively, with a 15-fold effect. The Kdissofmalonate is 0.66 0.05 mM and 0.08 0.01 mM, in theabsence and presence of ATP, respectively, with an 8.5-foldeffect. The affinity of ATP for SR significantly increases whenglycine is bound to the enzyme.Results indicate that drugs aimed at regulating SR activitymight be developed targeting both the active site and the ATPbinding site.