Novel phosphoserine phosphatase inhibitors

Abstract

Phosphoserine phosphatase (EC 3.1.1.3) catalyzes the final step in the major pathway of l-serine biosynthesis in brain. This enzyme may also regulate the levels of glycine and d-serine, the known and putative co-agonists for the glycine site of the N-methyl- d-aspartate receptor in caudal and rostral brain regions, respectively. Using l-phosphoserine as substrate, the rank order potency for inhibition of phosphoserine phosphatase was p-chloromercuriphenylsulfonic acid (CMPSA)>glycerophosphorylcholine≫hexadecylphosphocholine≥phosphorylcholine> N-ethylmaleimide≥ l-serine>fluoride> d-2-amino-3-phosphonopropionic acid ( d-AP3). Glycerylphosphorylcholine (IC 50 18 μM) was found to be an uncompetitive inhibitor of phosphoserine phosphatase. Glycerylphosphorylcholine probably binds a novel site on the enzyme since the known allosteric inhibitor l-serine is highly selective for its feedback regulatory site, indicated by the inactivity of 25 l-serine analogs. Fluoride ion (IC 50 770 μM) may bind the active site as has been shown for other Mg 2+-dependent enzymes. The sulfhydryl reagent CMPSA is a potent, noncompetitive inhibitor of the enzyme using l-phosphoserine as substrate (IC 50 9 μM) but is >300-fold less potent using d-phosphoserine as substrate. Substrate-dependent differences are also observed with the sulfhydryl alkylator N-ethylmaleimide, which inhibits l-phosphoserine, but stimulates d-phosphoserine hydrolysis. These sulfhydryl reagents may dissociate multimeric forms of the enzyme to form monomers; the multimeric forms and monomers may preferentially cleave l- and d-phosphoserine, respectively. Phosphorylcholine esters and sulfhydryl reagents may prove useful in determining the contribution of phosphoserine phosphatase to the biosynthesis of glycine and d-serine in neuronal tissue in vitro.