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

D. R. Brandt, E. M. Ross

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)266-272
Number of pages7
JournalJournal of Biological Chemistry
Volume260
Issue number1
StatePublished - 1985

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GTP Phosphohydrolases
Guanosine Triphosphate
Adenylyl Cyclases
Nucleotides
Guanylyl Imidodiphosphate
Enzymes
Hydrolysis
Magnesium Chloride
Guanine Nucleotides
Guanosine
GTP-Binding Proteins
Edetic Acid
Carrier Proteins
Hormones
Rabbits
Liver
Substrates
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{f36aa7acab10464d86b80fe33e356a3c,
title = "GTPase activity of the stimulatory GTP-binding regulatory of adenylate cyclase, G(s). Accumulation and turnover of enzyme-nucleotide intermediates",
abstract = "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.",
author = "Brandt, {D. R.} and Ross, {E. M.}",
year = "1985",
language = "English (US)",
volume = "260",
pages = "266--272",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
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T1 - GTPase activity of the stimulatory GTP-binding regulatory of adenylate cyclase, G(s). Accumulation and turnover of enzyme-nucleotide intermediates

AU - Brandt, D. R.

AU - Ross, E. M.

PY - 1985

Y1 - 1985

N2 - 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.

AB - 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.

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