Evidence for a phosphoryl-enzyme intermediate in phosphate ester hydrolysis by purple acid phosphatase from bovine spleen.
The possibility of the existence of a covalent enzyme-phosphoryl intermediate, E-PO3, during catalysis of phosphate ester hydrolysis by the purple acid phosphatase (PAP) from bovine spleen has been examined. Transphosphorylation experiments show that up to 22% of the phosphoryl group from p-nitrophenyl phosphate (PNPP) can be transferred to primary alcohols. Burst experiments at high pH (9.1 or 8.1 for reduced or oxidized PAP, respectively), where hydrolysis of a phosphoenzyme intermediate is expected to be rate-limiting, show clear evidence for stoichiometric bursts of p-nitrophenolate from PNPP. The formation of base-stable, acid-sensitive adducts between PAP and the 32PO3 group of [gamma-32P]ATP has been demonstrated. The pH dependence of the kinetics parameters for reduced PAP has been determined over the range pH 3-8; a feature with a pKa of approximately 6.75 that is attributable to the enzyme-substrate complex is observed. Taken together, the present results are consistent with a two-stem pseudo Uni Bi mechanism that utilizes a covalent enzyme-phosphoryl intermediate, possibly a phosphohistidine.
Department of Chemistry, University of Virginia, Charlottesville 22901.
J B
JB
Vincent
M W
MW
Crowder
B A
BA
Averill
D000135:Acid Phosphatase / Q000378:metabolism*; D000818:Animals; D000838:Anions; D001665:Binding Sites; D001692:Biological Transport; D002417:Cattle; D004952:Esters / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D009943:Organophosphorus Compounds / Q000378:metabolism*; D013154:Spleen / Q000201:enzymology*
D000838:Anions; D004952:Esters; D009943:Organophosphorus Compounds; D000135:Acid Phosphatase