Purine nucleoside phosphorylase
phosphate ion binding
Effect of the phosphate substrate on drug-inhibitor binding to human purine nucleoside phosphorylase.
The thermodynamics of the drug-inhibitors acyclovir, ganciclovir, and 9-benzylguanine binding to human purine nucleoside phosphorylase (hsPNP) were determined from isothermal titration calorimetry as a function of the substrate phosphate ion (Pi) concentration from 0 to 0.125 M and temperature from 15 degrees C to 35 degrees C. At 25 degrees C and with an increase in the Pi concentration from 0 to 50mM, acyclovir binding becomes more entropically-driven and ganciclovir binding becomes more enthalpically-driven. At 25 degrees C, the tighter 9-benzylguanine binding reaction goes from an enthalpically-driven reaction in the absence of Pi to an entropically-driven reaction at 10 mM Pi, and the enthalpically-driven nature of the binding reaction is restored at 75 mM Pi. Since the dependencies of the driving-nature of the binding reactions on Pi concentration can be simulated by Pi binding to its catalytic site, it is believed that bound Pi affects the interactions of the side-chains with the ribose catalytic site. However, the binding constants are unaffected by change in the bound Pi concentration because of enthalpy-entropy compensation. The enzymatic activity of hsPNP was determined by an ITC-based assay employing 7-methylguanosine and Pi as the substrates. The heat of reaction determined from the assay increased by 7.5 kJ mol(-1) with increase in Pi concentration from 50 to 100mM and is attributed to weak binding of the Pi to a secondary regulatory site. Although the binding constants of acyclovir and ganciclovir at 20 microM hsPNP were in agreement with the inverse inhibition constants determined from the ITC enzyme inhibition assays at 60 nM, the binding constant of 9-benzylguanine, which interacts with Phe159 from an adjacent subunit, decreased from 5.62 x 10(5) M(-1) to 1.14 x 10(5) M(-1). This reduction in the 9-benzylguanine binding affinity along with a 7-fold increase in the specific activity of hsPNP at 14.5 nM results from partial dissociation of the hsPNP trimer into monomers below the 60 nM level.