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Y. Ho; Probing the Catalytic Mechanism of Retinal cGMP Phosphodiesterase with Derterium Oxide Solvent Isotope Effect and Proton Inventory . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1522.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Retinal cGMP Phosphodiesterase plays an essential role in photoexcitation of rod cells. In this study, I investigate the catalytic and activation mechanism of PDE. Methods: The kinetics of cGMP hydrolysis catalyzed by PDE was studied with D2O solvent isotope effect and proton inventory methods. Results: The rate of cGMP hydrolysis catalyzed by trypsin- or transducin(T-Gpp(NH)p)activated PDE exhibited a D2O solvent isotope effect of 1.7 indicating that water molecule participated at the rate-limiting step of catalysis. The involvement of protons in catalysis was quantified by proton inventory method - by varying molar fraction of D2O. Trypsin-activated PDE exhibited a linear relationship on proton inventory analysis. This result is consistent with a general base catalyzed single proton transfer mechanism. On the other hand, T-Gpp(NH)p activated PDE showed a convex curve on proton inventory. This suggested that T-Gpp(NH)p activation of PDE was rate-limiting at low D2O content. However at high D2O concentration, The T-Gpp(NH)p:PDE complex was stablized that the cGMP hydrolysis reaction become dominant. The proton inventory result suggested that T-Gpp(NH)p is tightly associated with the PDE complex and the dissociation of inhibitory peptide of PDE is not an obligatory step during activation. Conclusions: The catalytic mechanism of retinal cGMP phosphodiesterase involves a general base catalyzed single proton transfer reaction. Transducin activation of PDE involves a tight complex including T-GTP, the PDE catalytic subnits and the inhibitory peptide.
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