Pre-Steady-State Kinetics of the Binding and Hydrolysis of mant-cGMP by Phosphodiesterase-5

Phosphodiesterase-5 (PDE5), receptor for the drugs sildenafil (Viagra  ), vardenafil (Levitra  ), and tadalafil (Cialis  ), selectively hydrolyzes the second messenger cGMP.However, its hydrolytic mechanism is unknown.The hydrolyzable substrate analog mant-cGMP and isolated PDE5 catalytic domain were used to study substrate interaction and subsequent hydrolysis.Stopped-flow analysis by Förster/fluorescence resonance energy transfer (PDE5 tryptophans mant-cGMP) identified five steps.The rate constant of mant-cGMP binding to PDE5 catalytic domain was measured to be 2.4 x 10 7 M -1 s -1 at 4C but was too rapid to be measured at 20 o C. Substrate binding was followed by two additional Mg 2+ -dependent, and mant-cGMP concentration-independent increases in fluorescence (Steps 2 and 3).The decrease in fluorescence that occurred with Step 4 had the same rate as the hydrolytic step measured by quench-flow and was attributed to be hydrolysis followed by rapid product dissociation.These results suggested that hydrolysis of mant-cGMP is the rate-limiting step of the enzymatic reaction and that the final slow increase in fluorescence is due to the binding of nonenzymatically isomerized product, mant-5'-GMP (2'3') to the enzyme.We propose a model whereby mant-cGMP in the presence of Mg 2+ rapidly binds to the catalytic site of the enzyme, followed by multi-step formation of increasingly tight enzyme-substrate complexes prior to mant-cGMP hydrolysis and rapid dissociation of the product, mant-5'-GMP.