Purine-nucleoside phosphorylase from Salmonella typhimurium and Escherichia coli. Initial velocity kinetics, ligand banding, and reaction mechanism

Abstract

Purine nucleoside phosphorylase from Salmonella typhimurium has been subjected to kinetic analysis i.e. determination of initial velocity patterns and product inhibition studies. The kinetic results suggest that the enzyme works by a sequential reaction mechanism, where the nucleoside, phosphate, and pentose 1-phosphate are all able to bind to the free enzyme, whereas it appears that the purine base binds after addition of the pentose 1-phosphate. The proposed mechanism is confirmed by substrate binding studies. In addition to the enzyme-substrate complexes suggested by the kinetics, the binding studies revealed a 'dead end' complex, consisting of enzyme, phosphate, and purine base. Similar binding experiments were carried out using the enzyme from Escherichia coli. The results suggest that this enzyme works by an identical reaction mechanism. The binding data are in agreement with the presence of six binding sites per native enzyme molecule, one binding site per subunit, for each ligand. Both enzymes show normal Michaelis-Menten kinetics for their substrates with the exception of phosphate, for which the double-reciprocal plots are concave down. This behaviour is seen in both binding and velocity curves, and most likely is a result of negative cooperativity in the binding of phosphate to the enzyme.