J. Biol. Chem., Vol. 267, Issue 11, 7342-7351, 04, 1992
Purine nucleoside phosphorylase. Kinetic mechanism of the enzyme from calf spleen
DJ Porter
Experimental Therapy Division, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
Ribose 1-phosphate, phosphate, and acyclovir diphosphate quenched the
fluorescence of purine nucleoside phosphorylase at pH 7.1 and 25 degrees C.
The fluorescence of enzyme-bound guanine was similar to that of anionic
guanine in ethanol. Guanine and ribose 1-phosphate bound to free enzyme,
whereas inosine and guanosine were not bound to free enzyme in the absence
of phosphate. Thus, synthesis proceeded by a random mechanism, and
phosphorolysis proceeded by an ordered mechanism. Steady-state kinetic data
for the phosphorolysis of 100 microM guanosine were fitted to a
bifunctional kinetic model with catalytic rate constants of 22 and 1.3 s-1.
The dissociation rate constants for guanine from the enzyme-guanine complex
at high and low phosphate concentrations were similar to the catalytic rate
constants. Fluorescence changes of the enzyme during phosphorolysis
suggested that ribose 1-phosphate dissociated from the enzyme ribose
1-phosphate- guanine complex rapidly and that guanine dissociated from the
enzyme- guanine complex slowly. The association and dissociation rate
constants for acyclovir diphosphate, a potent inhibitor of the enzyme
(Tuttle, J. V., and Krenitsky, T. A. (1984) J. Biol. Chem. 259, 4065-4069),
were also dependent on phosphate concentration. The effects of phosphate
are discussed in terms of a dual functional binding site for phosphate.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
