Reaction rate and collisional efficiency of the rhodopsin-transducin system in intact retinal rods

Abstract

A model of transducin activation is constructed from its partial reactions (formation of metarhodopsin II, association, and dissociation of the rhodopsin-transducin complex). The kinetic equations of the model are solved both numerically and, for small photoactivation, analytically. From data on the partial reactions in vitro, rate and activation energy profile of amplified transducin turnover are modeled and compared with measured light-scattering signals of transducin activation in intact retinal rods. The data leave one free parameter, the rate of association between transducin and rhodopsin. Best fit is achieved for an activation energy of 35 kJ/mol, indicating lateral membrane diffusion of the proteins as its main determinant. The absolute value of the association rate is discussed in terms of the success of collisions to form the catalytic complex. It is greater than 30% for the intact retina and 10 times lower after permeabilization with staphylococcus aureus alpha-toxin. Dissociation rates for micromolar guanosinetriphosphale (GTP) (Kohl, B., and K. P. Hofmann, 1987. Biophys. J. 52:271-277) must be extrapolated linearly up to the millimolar range to explain the rapid transducin turnover in situ. This is interpreted by an unstable rhodopsin-transducin-GTP transient state. At the time of maximal turnover after a flash, the rate of activation is determined as 30, 120, 800, 2,500, and 4,000 activated transducins per photoactivated rhodopsin and second at 5, 10, 20, 30, 37 degrees C, respectively.

Pre-tRNA turnover catalyzed by the yeast nuclear RNase P holoenzyme is limited by product release

“…Curved Arrhenius plots with large energies of activation (E a > 40 kcal/mol) at lower temperatures have been observed previously for the kinetics of complex reactions, such as actin filaments moving with respect to myosin (Anson 1992), rhodopsin activation (Kahlert and Hofmann 1991), and formation of E. coli RecA protein filaments (Wilson and Benight 1990). In addition, nonlinear Arrhenius plots with similar activation energies (z40-50 kcal/mol) have been observed previously for association kinetics for nucleic acids and protein-nucleic acid complexes, in-cluding the migration of DNA cruciforms (Lilley 1985), association of E. coli s 70 RNA polymerase with the lP R promoter (Roe et al 1985;Saecker et al 2002), E. coli RecA protein binding to duplex DNA (Pugh and Cox 1988), and substrate binding to the hammerhead ribozyme (Peracchi 1999) or to a small deoxyribozyme (Bonaccio et al 2004).…”

A quantitative account of the activation steps involved in phototransduction in amphibian photoreceptors.

“…ca 4200/6300). We therefore conclude that it is likely that each of these protein activation steps proceeds at a rate that cannot be far short of its diffusion limit; see also Kahlert & Hofmann (1991).…”

Fluorescence/Bioluminescence Resonance Energy Transfer Techniques to Study G-Protein-Coupled Receptor Activation and Signaling

“…On the other hand, activation of rhodopsin, the best-studied class A GPCR, is well documented to occur much faster. In fact, activation of the light receptor rhodopsin can be observed within 1 ms of light triggering (Kahlert and Hofmann, 1991;Pugh and Lamb, 1993;Makino et al, 2003), and even the downstream closure of the cGMP-gated cation channel-which requires the intermediate steps of transducin activation and cGMP hydrolysis by the transducin-activated phosphodiesterase-is observed within 200 ms (Makino et al, 2003). Thus, it is obvious that GPCR activation can, in principle, occur much faster than observed so far with GPCR FRET sensors.…”