GTPase activity of the stimulatory GTP-binding regulatory of adenylate cyclase, G(s). Accumulation and turnover of enzyme-nucleotide intermediates

The GTPase activity of the stimulatory guanine nucleotide-binding regulatory protein (G(s)) of hormone-sensitive adenylate cyclase was investigated using purified rabbit hepatic G(s) and either [α-³²P]- or [γ-³²P]GTP as substrate. The binding of [³⁵S]guanosine 5'-O-(thiotriphosphate) (GTPγS) was used to quantitate the total concentration of G(s). 1) GTPase activity was a saturable function of the concentration of GTP, with K(m) = 0.3 μM. MgCl₂ monotonically increased the activity. The maximum observed turnover number was about 1.5 min^-1. 2) During steady-state hydrolysis, 20-40% of total G(s) could be trapped as a G(s)-GDP complex and 1-2% could be trapped as G(s)-GTP. The hydrolysis of G(s)-GTP to G(s)-GDP occurred with t( 1/2 ) ≤ 5 s at 30°C and t( 1/2 ) ~ 1 min at 0°C. Hydrolysis of G(s)-GTP was inhibited by 1.0 mM EDTA in the absence of added Mg²⁺. 3) The rate of formation of G(s)-GDP and the initial GTPase rate varied in parallel as functions of the concentrations of either GTP or MgCl₂ (above 0.1 mM Mg²⁺). The ratio of the rate of accumulation of G(s)-GDP to the GTPase rate was constant at 0.3-0.4. 4) The rate of dissociation of assayable G(s)-GDP was biphasic. The initial phase accounted for 60-80% of total assayable G(s)-GDP and was characterized by a t( 1/2 ) of about 1 min. 5) Lubrol 12A9 potently inhibited the GTPase reaction and the dissociation of G(s)-GDP in parallel, and inhibition of product release may account for the inhibition of steady-state hydrolysis. 6) The β and γ subunits of G(s) markedly inhibited the dissociation of GDP from G(s) in contrast to their ability to stimulate the dissociation of GTPγS. 7) GDP, GTPγS, and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) competitively inhibited the accumulation of G(s)-GDP. GTPγS and Gpp(NH)p inhibited the GTPase reaction noncompetitively, GDP displayed mixed inhibition, and P(i) did not inhibit. These data are interpretable in terms of the coexistence of two specific mechanistic pathways for the overal GTPase reaction.