Abstract
Rabbit liver purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase EC 2.4.2.1.) was purified to homogeneity by column chromatography and ammonium sulfate fractionation. Homogeneity was established by disc gel electrophoresis in presence and absence of sodium dodecyl sulfate, and isoelectric focusing. Molecular weights of 46,000 and 39,000 were determined, respectively, by gel filtration and by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Product inhibition was observed with guanine and hypoxanthine as strong competitive inhibitors for the enzymatic phosphorolysis of guanosine. Respective Kis calculated were 1.25 x 10(-5) M for guanine and 2.5 x 10(-5) M for hypoxanthine. Ribose 1-phosphate, another product of the reaction, gave noncompetitive inhibition with guanosine as variable substrate, and an inhibition constant of 3.61 x 10(-4) M was calculated. The protection of essential --SH groups on the enzyme, by 2-mercaptoethanol or dithiothreitol, was necessary for the maintenance of enzyme activity. Noncompetitive inhibition was observed for p-chloromercuribenzoate with an inhibition constant of 5.68 x 10(-6)M. Complete reversal of this inhibition by an excess of 2-mercaptoethanol or dithiothreitol was demonstrated. In the presence of methylene blue, the enzyme showed a high sensitivity to photooxidation and a dependence of photoinactivation on pH, strongly implicating histidine as the susceptible group at the active site of the enzyme. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 5.5 and pH 8.5 The chemical and kinetic evidences suggest that histidine and cysteine may be essential for catalysis. Inorganic orthophosphate (Km 1.54 x 10(-2) M) was an obligatory anion requirement, and arsenate substituted for phosphate with comparable results. Guanosine (Km 5.00 x 10(-5) M), deoxyguanosine (Km 1.00 x 10(-4)M) and inosine (Km 1.33 x 10(-4)M), were substrates for enzymatic phosphorolysis. Xanthosine was an extremely poor substrate, and adenosine was not phosphorylyzed at 20-fold excess of the homogeneous enzyme. Guanine (Km 1.82 x 10(-5)M),ribose 1-phosphate (Km 1.34 x 10(-4) M) and hypoxanthine were substrates for the reverse reaction, namely, the enzymatic synthesis of nucleosides. The initial velocity studies of the saturation of the enzyme with guanosine, at various fixed concentrations of inorganic orthophosphate, suggest a sequential bireactant catalytic mechanism for the enzyme.
Highlights
Inhibition by ribose l-phosphate was noncompetitive for the phosphorolysis of guanosine, and an inhibition constant of 3.61 x lo-’ M was calculated (Fig. 4)
The limiting concentration of inorganic orthophosphate for optimal affinity of the enzyme for guanosine was determined at 20 mM (Fig. 10)
The initial velocity pattern observed for the phosphorolysis of guanosine, at various fixed concentrations of inorganic orthophosphate, suggests that the catalytic mechanism of rabbit liver purine nucleoside phosphorylase is a sequential bireactant mechanism [26]
Summary
Frozen rabbit livers were purchased from Pel-Freez Bio-Animals (Rogers, Ark.). DEAE-cellulose (0.9 meq/g) and hydroxylapatite were purchased from Bio-Rad laboratories (Richmond, Calif.). The molecular weight of the enzyme was calculated from a plot of elution volume versus log molecular weight, after the method of Andrews [24]; and (b) sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis after the method of Weber and Osborn [19]. 85% of the adsorbed proteins were eluted from the column within 2 liters of 0.05 M phosphate buffer (pH 7.5), with no observable enzyme activity. The precipitated enzyme was recovered by centrifugation, reconstituted in 0.05 M phosphate buffer (pH 7.5), and dialyzed for 2 hours against running tap water. Step VIII: Second DEAE-cellulose Chromatography-The dialyzed enzyme (5.0 ml containing 13.1 mg of protein) was adsorbed on a column of DEAE-cellulose (1.6 cm x 10.0 cm), and eluted with 0.025 M citrate buffer (pH 6.8). At this stage of the purification, a minor contamination by a single protein was observed by polyacrylamide disc electrophoresis of the enzyme
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