**4. G protein activation kinetics**

Rhodopsin and related receptors **catalyze** G protein activation (Hamm, 1998): this means that each receptor sequentially activates several G proteins by facilitating the release of GDP, thereby allowing GTP binding. Rhodopsin does not enter in one of the Enzyme Commission "E.C." subclasses, as it does not catalyze the rupture or formation of covalent bond(s). The equations describing the reaction kinetics are nevertheless identical to those describing "ping pong" (double displacement) enzyme reaction (Waelbroeck *et al.*, 1997; Heck and Hofmann, 2001; Ernst *et al.*, 2007): G protein binding (substrate 1) is followed by GDP release (product 1), and GTP binding (substrate 2) is followed by the release of the activated G protein (product 2).

The kinetics of transducin activation by rhodopsin have been analyzed in detail (Heck and Hofmann, 2001; Ernst *et al.*, 2007). Rhodopsin recognizes transiently the GDP-bound trimeric G protein, transducin, and activates transducin at the diffusion limit (Ernst *et al.*, 2007). The physiological concentrations of the two "substrates" (GDP-bound transducin and GTP) are close to their respective Michaelis constants, KM. In the case of double displacement reactions, it is unfortunately impossible to the individual rate constants of each reaction from the kinetic data (KM, Vmax). The [Substrate]/KM ratios at physiological concentrations

Fig. 6. Ribbon representation of Gβγ showing the interaction surface with Gα or with effectors. Ribbon representation of Gβγ in different crystallized complexes – the Gβ (blue) and Gγ (green) subunits only are shown for simplicity. The amino acids that belong to the Gα recognition surface (PDB ref 2BCG: top), to the phosducin binding site (1GP2: bottom left) or to the kinase, GRK2 binding surface (bottom right : 1OMW) are shown as space

Rhodopsin and related receptors **catalyze** G protein activation (Hamm, 1998): this means that each receptor sequentially activates several G proteins by facilitating the release of GDP, thereby allowing GTP binding. Rhodopsin does not enter in one of the Enzyme Commission "E.C." subclasses, as it does not catalyze the rupture or formation of covalent bond(s). The equations describing the reaction kinetics are nevertheless identical to those describing "ping pong" (double displacement) enzyme reaction (Waelbroeck *et al.*, 1997; Heck and Hofmann, 2001; Ernst *et al.*, 2007): G protein binding (substrate 1) is followed by GDP release (product 1), and GTP binding (substrate 2) is followed by the release of the

The kinetics of transducin activation by rhodopsin have been analyzed in detail (Heck and Hofmann, 2001; Ernst *et al.*, 2007). Rhodopsin recognizes transiently the GDP-bound trimeric G protein, transducin, and activates transducin at the diffusion limit (Ernst *et al.*, 2007). The physiological concentrations of the two "substrates" (GDP-bound transducin and GTP) are close to their respective Michaelis constants, KM. In the case of double displacement reactions, it is unfortunately impossible to the individual rate constants of each reaction from the kinetic data (KM, Vmax). The [Substrate]/KM ratios at physiological concentrations

filling structures, in yellow.

**4. G protein activation kinetics**

activated G protein (product 2).

nevertheless strongly suggest that the concentrations of the four reaction intermediates, Rh\*, Rh\*-GGDP, Rh\*-G and Rh\*-GGTP (where Rh\* is the light activated rhodopsin) are similar (Roberts and Waelbroeck, 2004): none of the reaction intermediates accumulates. These characteristics are reminiscent of the properties of triose phosphate isomerase and other "kinetically perfect enzymes" (Albery and Knowles, 1976): all the reaction intermediates have very similar free energies at physiological substrate concentrations and the energy barriers separating the different enzyme states are very low, thereby allowing the reaction to proceed at the diffusion limit.

Enzymes accelerate reactions by stabilizing the "transition state", that is, the state with the highest energy along the reaction coordinates. Trimeric G proteins cannot be purified in the absence of guanyl nucleotides: they are unstable when empty. As explained below, agonists stabilize the agonist-receptor-G protein ternary complex (that includes an empty G protein): like enzymes, active GPCRs catalyze G protein activation by decreasing the free energy of the transition state, that is, the empty G protein (Waelbroeck, 1999). GTP recognition by the G protein destabilizes the ternary complex: this induces activated G protein release - and allows the catalytic activation of several G proteins by a single receptor.
