Kinetic control of guanine nucleotide binding to soluble Gαq

Binding of guanine nucleotides to heterotrimeric G proteins is controlled primarily by kinetic factors, such as the release of bound GDP, rather than by affinity alone. Detergent-solubilized G alpha(q) displays unusual guanine nucleotide binding properties in comparison with other G protein a subunits. Under conditions where most G proteins bind nearly stoichiometric GTP gamma S in 5-30 min at micromolar nucleotide concentrations, GTP gamma S binding to G alpha(q) is slow (>1 hr to completion), markedly substoichiometric, and dependent upon high concentrations of nucleotide (0.1 to 0.2 mM). Although the latter two properties suggest low affinity, GTP gamma S dissociation is immeasurably slow under commonly used conditions. We found that purified G alpha(q) can bind stoichiometric GTP gamma S, but that binding is controlled kinetically by a combination of factors. GDP (or IDP) dissociated slowly from G alpha(q), but the dissociation rate increased linearly with the concentration of (NH4)(2)SO4 up to 0.75 M (similar to 20-fold acceleration). The resulting GDP-free G alpha(q) was labile to rapid and irreversible denaturation, however (rate constant greater than or equal to 1 min(-1) at 20 degrees). Denaturation competed kinetically with relatively slow GTP gamma S association, such that stoichiometric binding was only attained at 100 mu M GTP gamma S. These findings reconcile the slowly reversible binding of GTP gamma S to G alpha(q) with the other behaviors that suggested lower affinity, and point out that events subsequent to GDP dissociation can markedly influence the rates and extents of guanine nucleotide binding to G protein alpha subunits. Understanding these interactions allowed the direct, accurate quantitation of active G alpha(q) by a simple GTP gamma S binding assay in the presence of (NH4)(2)SO4, and similarly can prevent underestimation of the concentrations of other G proteins. BIOCHEM PHARMACOL 58;1: 39-48, 1999. (C) 1999 Elsevier Science Inc.