Pre-Steady-State Kinetics of the Binding and Hydrolysis of mant-cGMP by Phosphodiesterase-5
Journal: International Journal of Biochemistry & Physiology (Vol.1, No. 1)Publication Date: 2016-06-21
Authors : Morris GZ Ke H Wang H Francis SH Corbin JD Belknap B; White HD;
Page : 1-12
Keywords : Phosphodiesterase-5 (PDE5); mant-cGMP; Pre-steady-state; TLC; FRET;
Abstract
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 107 M-1 s -1 at 4C but was too rapid to be measured at 20oC. Substrate binding was followed by two additional Mg2+-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 Mg2+ 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.
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