title: Evidence for a phosphoenzyme intermediate formed during catalysis by pyridoxal phosphatase from human erythrocytes.
abstract: 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 [32P]PLP, followed by quenching in acid, resulted in trapping 0.14-0.24 mol of 32P per mol of subunit. The incorporation of 32P was not due to Schiff base formation. Phosphorylation of the enzyme by [32P]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 Pi 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 32P-labeled phosphatase was digested with pepsin, and two radioactive peaks were isolated by reversed-phase chromatography. However, definitive sequences were not obtained.
authors.affiliation: Department of Biochemistry, University of Louisville School of Medicine, Kentucky 40292.
authors.forename: G J
authors.initials: GJ
authors.lastname: Gao
authors.forename: M L
authors.initials: ML
authors.lastname: Fonda
mesh_terms: D002384:Catalysis; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000097:blood*; D011732:Pyridoxal Phosphate / Q000378:metabolism
chemical_list: D010710:Phosphates; D010750:Phosphoproteins; D011732:Pyridoxal Phosphate; C022493:pyridoxal-5-phosphatase; D010744:Phosphoric Monoester Hydrolases