Bovine brain purine-nucleoside phosphorylase purification, characterization, and catalytic mechanism.
Bovine brain purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) was purified to homogeneity at a specific activity of 78 mumol min-1 mg of protein-1. A molecular weight of 78 000-80 000 was calculated for the native enzyme by fel filtration on Sephadex. Gel electrophoresis in the presence of sodium dodecyl sulfate indicated subunits of molecular weight of 38 000. Chemical and kinetic studies strongly implicated histidine and cysteine as catalytic groups at the active site of the enzyme. The pKa's determined for ionizable groups at the active site of the free enzyme were 5.8 and 8.2. Enzyme completely inactivated by p-chloromercuribenzoate was partially reactivated enzyme. A strong susceptibility to photooxidation in presence of methylene blue was observed. Photoinactivation was pH dependent, implicating histidine as the susceptible group at the active site. A rapid loss of catalytic activity upon incubation at 55 degrees C suggested heat lability. An activation energy of 9.6 kcal/mol was calculated. The nature of the catalytic mechanism of the enzyme was investigated, and initial velocity studies showed linear converging patterns of double-reciprocal plots of the data, consistent with a sequential catalytic mechanism. The product inhibition pattern was at variance with both the ordered Bi-Bi and random mechanisms. The observed competition between purine and nucleoside, and between inorganic orthophosphate and ribose 1-phosphate for this ordered mechanism, suggest a Theorell-Chance mechanism. Michaelis constants determined for substrates of the enzyme were 4.35 X 10(-5) M for guanosine, 3.00 X 10(-5) M for guanine, and 2.15 X 10(-2) M for inorganic orthophosphate.
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D011683:Purine-Nucleoside Phosphorylase / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship
D011683:Purine-Nucleoside Phosphorylase