JBC, Vol. 251, Issue 2, 407-413, Jan, 1976
Monomeric purine nucleoside phosphorylase from rabbit liver. Purification and characterization
A. S. Lewis and M. D. Glantz
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.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
