Synthesis in Escherichia coli of GTPase-deficient mutants of Gsα

作者：M P Graziano, Alfred G. Gilman · 发表于：Journal of Biological Chemistry · 年份：1989 · DOI：10.1016/s0021-9258(19)84854-8 · 被引用次数：259 · 研究领域：Protein Kinase Regulation and GTPase Signaling、Receptor Mechanisms and Signaling、Cellular transport and secretion

We have reduced the GTPase activity of the alpha subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase, by introduction of point mutations analogous to those described in p21ras. Mutants G49V and Q227L differ from the wild type protein in the substitution of Val for Gly49 and Leu for Gln227, respectively (analogous to positions 12 and 61 in p21ras). Wild type and mutant proteins were synthesized in Escherichia coli, purified, and characterized. The rate constants for dissociation of GDP from G49V recombinant Gs alpha (rGs alpha) (0.47/min) and Q227L rGs alpha (0.23/min) differ by no more than 2-fold from that observed for the wild type protein (0.5/min). In marked contrast, the rate constants for hydrolysis of GTP by G49V rGs alpha (0.78/min) and Q227L rGs alpha (0.03-0.06/min) are 4-fold and roughly 100-fold slower than that for wild type rGs alpha (3.5/min). These reductions in the rate of hydrolysis of GTP result in significant fractional occupancy of these proteins by GTP in the presence of the nucleotide, 0.37 for G49V rGs alpha and 0.78 for Q227L rGs alpha, compared to 0.05 for wild type rGs alpha. When reconstituted with cyc- (Gs alpha-deficient) S49 cell membranes or purified adenylyl cyclase, both mutant proteins stimulate adenylyl cyclase activity in the presence of GTP to a much greater extent than does wild type Gs alpha; their maximal ability to activate the enzyme is largely unaltered. The fractional ability of a given...