Purine nucleoside phosphorylase kinetics

The purine nucleoside phosphorylases (PNPs) are N-ribosyltransferases (Figure 7-7) where transition state analogue design on the basis of kinetic isotope effects analysis has had success. The inhibition of phosphorylation catalyzed by human... 

For example, intact Ehrlich ascites tumor cells, or extracts therefrom, transfer the ribosyl group of uridine to hypoxanthine and thereby catalyze the net synthesis of inosine this reaction depends upon the coupled actions of uridine phosphorylase and purine nucleoside phosphorylase (89). Similar ribosyl transfers have been demonstrated with bacterial cells and extracts. Krenitsky has studied the kinetics of exchange between uracil-2- C and nonisotopic uridine catalyzed by highly purified uridine phosphorylase (30) ... 

Table 2. Kinetic studies of erythrocyte purine nucleoside phosphorylase...

The ability to accurately compute kinetic isotope effects (KIEs) for chemical reactions in solution and in enzymes is important because the measured KIEs provide the most direct probe to the nature of the transition state and the computational results can help rationalize experimental findings. This is illustrated by the work of Schramm and co-workers, who have used the experimental KIEs to develop transition state models for the enzymatic process catalyzed by purine nucleoside phosphorylase (PNP), which in turn were used to design picomolar inhibitors. In principle, Schramm s approach can be applied to other enzymes however, in order to establish a useful transition state model for enzymatic reactions, it is often necessary to use sophisticated computational methods to model the structure of the transition state and to match the computed KIEs with experiments. The challenge to theory is the difficulty in accurately determining the small difference in free energy of activation due to isotope replacements, especially for secondary and heavy isotope effects. Furthermore, unlike studies of reactions in the gas phase, one has to consider... 

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