Different kinetic properties of the two mutants, RAS2Ile152 and RAS2Val19, that suppress the CDC25 requirement in RAS/adenylate cyclase pathway in Saccharomyces cerevisiae.
The properties of RAS2Gly19----Val and RAS2Thr152----Ile, two mutants suppressing the CDC25 requirement for the activation of adenylate cyclase in Saccharomyces cerevisiae, were compared with the properties of wild-type RAS2. We examined (a) the guanine nucleotide interaction, (b) the intrinsic GTPase (EC 3.6.1-) activity, and (c) the ability to activate adenylate cyclase in vitro. The low GTPase of RAS2Val19 is associated with an increased stability of the GTP complex. By contrast, RAS2Ile152 shows a strong destabilization of the GDP complex (the dissociation rate constants of the RAS2Ile152.GDP complex is enhanced almost 50 times) and an increased GTPase activity. Remarkably, all the parameters of the interaction with GDP and GTP as well as the catalytic activity are modified by the two mutations in an opposite manner. Our kinetic results show that the functional modifications of RAS2 compensating for the CDC25 inactivation can not only be associated with the presence of a long-lived RAS2.GTP complex, but also with a rapid GDP to GTP exchange reaction. As a striking result, the functional modifications induced by Thr152----Ile activate the adenylate cyclase in vitro much more efficiently than those induced by Gly19----Val. This stresses the importance of a rapid regeneration of the RAS2.GTP complex for the activation of the adenylate cyclase pathway.
Créchet
J B
JB
Laboratoire de Biochimie, URA 240 du Centre National de la Recherche Scientifique, Ecole Polytechnique, Palaiseau, France.
Poullet
P
P
Camonis
J
J
Jacquet
M
M
Parmeggiani
A
A
D000262:Adenylyl Cyclases / Q000378:metabolism*; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D007700:Kinetics; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D018631:ras Proteins; D020666:ras-GRF1
C542351:CDC25 protein, S cerevisiae; D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D020666:ras-GRF1; D019204:GTP-Binding Proteins; C489015:RAS2 protein, S cerevisiae; D018631:ras Proteins; D000262:Adenylyl Cyclases