ISSN: 2577-4360
Authors: Morris GZ*, Ke H, Wang H, Francis SH, Corbin JD, Belknap B and White HD
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 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 at 4C but was too rapid to be measured at 20C. Substrate binding was followed by two additional Mg 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 to the enzyme. We propose a model whereby mant-cGMP in the presence of Mg 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.
Keywords: Phosphodiesterase-5 (PDE5); mant-cGMP; Pre-steady-state; TLC; FRET
