EVIDENCE FOR A PHOSPHOENZYME INTERMEDIATE FORMED DURING CATALYSIS BY PYRIDOXAL PHOSPHATASE FROM HUMAN ERYTHROCYTES

Pyridoxal phosphatase purified from human erythrocytes catalyzes the dephosphorylation of pyridoxal phosphate (PLP) and pyridoxine phosphate. The enzyme had phosphotransferase activity and transferred 20-25% of the phosphoryl group from either substrate to ethanol. Incubation of the enzyme with [P-32]PLP, followed by quenching in acid, resulted in trapping 0.14-0.24 mol of P-32 per mol of subunit. The incorporation of P-32 was not due to Schiff base formation. Phosphorylation of the enzyme by [P-32]PLP required catalysis by the enzyme and did not occur in the presence of excess pyridoxine phosphate or with denatured enzyme. The phosphoenzyme intermediate was relatively acid stable and very labile at high pH or in the presence of hydroxylamine. Woodward's reagent K, which specifically modifies acidic amino acid residues, inactivated the phosphatase in a concentration- and time-dependent manner which followed pseudo-first-order kinetics. Substrates or P-i protected the enzyme from inactivation. It is concluded that PLP phosphatase catalyzes the hydrolysis of PLP by forming a covalent phosphoenzyme intermediate and the intermediate may be an acylphosphate. The P-32-labeled phosphatase was digested with pepsin, and two radioactive peaks were isolated by reversed-phase chromatography. However, definitive sequences were not obtained. (C) 1994 Academic Press, Inc.