Allosteric behavior in transducin activation mediated by rhodopsin. Initial rate analysis of guanine nucleotide exchange
Wessling-Resnick, M.; Johnson, G.L.
Journal of Biological Chemistry 262(8): 3697-3705
1987
ISSN/ISBN: 0021-9258 PMID: 3102494 Accession: 004699467
Photolyzed rhodopsin acts in a catalytic manner to mediate the exchange of GTP for GDP bound to transducin. We have analyzed the steady-state kinetics of this activation process in order to determine the molecular mechanism of interactions between rhodopsin, transducin, and guanine nucleotides. Initial velocities (V0) of the exchange reaction catalyzed by rhodopsin were measured for various transducin concentrations at several fixed levels of the GTP analog, [35S]guanosine 5'-(3-O-thio)triphosphate (GTP.gamma.S). The initial rate data analysis rigorously demonstrates that rhodopsin mediates the activation of transducin by a double-displacement catalytic mechanism. the Michaelis-Menten curves determined as a function of [transducin] reveal remarkable allosteric behavior; analysis of this data yields a Hill coefficient of 2. Lineweaver-Burk plots of V0-1 versus (transducin]-1 display curvilinearity indicative of positive cooperativity and a series of parallel lines are generated by plotting V0-1 as a function of [transducin]-2. The plots of V0-1 versus [GTP.gamma.S]-1 show no evidence of allosterism and are a parallel series. Furthermore, the allosteric behavior observed in the activation of transducin is also witnessed in the rhodopsin-catalyzed guanine nucleotide exchange of the G protein's purified .alpha. subunit in the absence of the .beta.cntdot.gamma. subunit complex. The latter observation implies that the molecular basis for allosterism in the activation process residues in the interactions between the photoreceptor and transducin's .alpha. subunit.