The Catalytic Mechanism of Mammalian Adenylyl Cyclase
1997; Elsevier BV; Volume: 272; Issue: 44 Linguagem: Inglês
10.1074/jbc.272.44.27787
ISSN1083-351X
AutoresCarmen Dessauer, Alfred G. Gilman,
Tópico(s)Peptidase Inhibition and Analysis
ResumoThe mechanism of P-site inhibition of adenylyl cyclase has been probed by equilibrium binding measurements using 2′-[3H]deoxyadenosine, a P-site inhibitor, and by kinetic analysis of both the forward and reverse reactions (i.e. cyclic AMP and ATP synthesis, respectively). There is one binding site for 2′-deoxyadenosine per C1/C2 heterodimer; the K d is 40 ± 3 μm. Binding is observed only in the presence of one of the products of the adenylyl cyclase reaction, pyrophosphate (PPi). A substrate analog, Ap(CH2)pp (α,β-methylene adenosine 5′-triphosphate), and cyclic AMP compete for the P-site in the presence of PPi, but P-site analogs do not compete for substrate binding (in the absence of PPi). Kinetic analysis indicates that release of products from the enzyme is random. These facts permit formulation of a model for the adenylyl cyclase reaction, for which we provide substantial kinetic support. We propose that P-site analogs act as dead-end inhibitors of product release, stabilizing an enzyme-product (E-PPi) complex by binding at the active site. Although product release is random, cyclic AMP dissociates from the enzyme preferentially. Release of PPiis slow and partially rate-limiting. The mechanism of P-site inhibition of adenylyl cyclase has been probed by equilibrium binding measurements using 2′-[3H]deoxyadenosine, a P-site inhibitor, and by kinetic analysis of both the forward and reverse reactions (i.e. cyclic AMP and ATP synthesis, respectively). There is one binding site for 2′-deoxyadenosine per C1/C2 heterodimer; the K d is 40 ± 3 μm. Binding is observed only in the presence of one of the products of the adenylyl cyclase reaction, pyrophosphate (PPi). A substrate analog, Ap(CH2)pp (α,β-methylene adenosine 5′-triphosphate), and cyclic AMP compete for the P-site in the presence of PPi, but P-site analogs do not compete for substrate binding (in the absence of PPi). Kinetic analysis indicates that release of products from the enzyme is random. These facts permit formulation of a model for the adenylyl cyclase reaction, for which we provide substantial kinetic support. We propose that P-site analogs act as dead-end inhibitors of product release, stabilizing an enzyme-product (E-PPi) complex by binding at the active site. Although product release is random, cyclic AMP dissociates from the enzyme preferentially. Release of PPiis slow and partially rate-limiting. Adenosine and various analogs of the nucleoside have both stimulatory and inhibitory effects on adenylyl cyclase activity (reviewed in Ref. 1Londos C. Wolff J. Cooper D.M.F. Bar H.P. Drummand G.I. Physiological and Regulatory Functions of Adenosine and Adenine Nucleotides. Raven Press, New York1979: 271-281Google Scholar). Londos and Wolff (2Londos C. Wolff J. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5482-5486Crossref PubMed Scopus (489) Google Scholar) categorized these effects mechanistically, based on their structure-activity relationships. Two types of adenosine-reactive sites were identified: those with strict requirements for the ribose moiety, designated R sites, and those with strict structural constraints for interaction with the purine ring, designated P sites. R sites are the ligand-binding sites of adenosine-specific G protein 1The abbreviations used are: G protein, heterotrimeric guanine nucleotide-binding protein; Gsα, the α subunit of the G protein that stimulates adenylyl cyclase; Ap(CH2)pp, α,β-methylene adenosine 5′-triphosphate; GTPγS, guanosine 5′-(γ-thio)triphosphate; PPi, pyrophosphate. -coupled receptors, which can either stimulate or inhibit adenylyl cyclase activity indirectly, while P sites, whose occupancy inhibits cyclic AMP synthesis, are structural features of adenylyl cyclases themselves (2Londos C. Wolff J. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5482-5486Crossref PubMed Scopus (489) Google Scholar, 3Londos C. Preston M.S. J. Biol. Chem. 1977; 252: 5957-5961Abstract Full Text PDF PubMed Google Scholar, 4Johnson R.A. Saur W. Jakobs K.H. J. Biol. Chem. 1979; 254: 1094-1101Abstract Full Text PDF PubMed Google Scholar, 5Wolff J. Londos C. Cooper D.M.F. Adv. Cyclic Nucleotide Res. 1981; 14: 199-214PubMed Google Scholar, 6Londos C. Cooper D.M.F. Wolff J. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2551-2554Crossref PubMed Scopus (951) Google Scholar, 7Florio V.A. Ross E.M. Mol. Pharmacol. 1983; 24: 195-202PubMed Google Scholar). The physiological significance of P-site inhibition is unclear, but concentrations of 3′-AMP found in vivo appear sufficient to inhibit adenylyl cyclase activity (8Bushfield M. Shoshani I. Johnson R.A. Mol. Pharmacol. 1990; 38: 848-853PubMed Google Scholar). P-site inhibition is typically noncompetitive or uncompetitive with respect to substrate ATP, depending on the divalent cation utilized in the assay (Mn2+ usually yielding noncompetitive kinetics; Mg2+ uncompetitive) (3Londos C. Preston M.S. J. Biol. Chem. 1977; 252: 5957-5961Abstract Full Text PDF PubMed Google Scholar, 4Johnson R.A. Saur W. Jakobs K.H. J. Biol. Chem. 1979; 254: 1094-1101Abstract Full Text PDF PubMed Google Scholar, 9Weinryb I. Michel I.M. Biochim. Biophys. Acta. 1974; 334: 218-225Crossref Scopus (42) Google Scholar, 10Wolff J. Londos C. Cook G.H. Arch. Biochem. Biophys. 1978; 191: 161-168Crossref PubMed Scopus (35) Google Scholar, 11Welton A.F. Simko B.A. Biochim. Biophys. Acta. 1980; 615: 252-261Crossref PubMed Scopus (8) Google Scholar). Furthermore, the apparent potency of such inhibitors increases when adenylyl cyclase is activated (3Londos C. Preston M.S. J. Biol. Chem. 1977; 252: 5957-5961Abstract Full Text PDF PubMed Google Scholar, 4Johnson R.A. Saur W. Jakobs K.H. J. Biol. Chem. 1979; 254: 1094-1101Abstract Full Text PDF PubMed Google Scholar, 5Wolff J. Londos C. Cooper D.M.F. Adv. Cyclic Nucleotide Res. 1981; 14: 199-214PubMed Google Scholar, 7Florio V.A. Ross E.M. Mol. Pharmacol. 1983; 24: 195-202PubMed Google Scholar, 11Welton A.F. Simko B.A. Biochim. Biophys. Acta. 1980; 615: 252-261Crossref PubMed Scopus (8) Google Scholar). Representative P-site reagents, ordered by potency, include 2′,5′-dideoxy-3′-ATP > 2′,5′-dideoxy-3′-ADP > 2′,5′-dideoxy-3′-AMP > 2′-deoxy-3′-AMP > 3′-AMP > 2′-deoxyadenosine > adenosine (12Johnson R.A. Yeung S.-M.H. Stübner D. Bushfield M. Shoshani I. Mol. Pharmacol. 1989; 35: 681-688PubMed Google Scholar, 13Desaubry L. Shoshani I. Johnson R.A. J. Biol. Chem. 1996; 271: 2380-2382Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Although several mechanisms for P-site inhibition have been proposed, there is as yet no conclusive evidence to support any particular hypothesis. Using engineered, soluble forms of mammalian adenylyl cyclase, we and others have shown that the conserved cytosolic domains of the enzymes contain the structural components necessary for Gsα- and forskolin-stimulated adenylyl cyclase activity, as well as the characteristic features of P-site inhibition (14Tang W.-J. Gilman A.G. Science. 1995; 268: 1769-1772Crossref PubMed Scopus (165) Google Scholar, 15Dessauer C.W. Gilman A.G. J. Biol. Chem. 1996; 271: 16967-16974Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 16Whisnant R.E. Gilman A.G. Dessauer C.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6621-6625Crossref PubMed Scopus (117) Google Scholar, 17Yan S.-Z. Hahn D. Huang Z.-H. Tang W.-J. J. Biol. Chem. 1996; 271: 10941-10945Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 18Sunahara R.K. Dessauer C.W. Whisnant R.E. Kleuss C. Gilman A.G. J. Biol. Chem. 1997; 272: 22265-22271Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). We have also utilized the competitive substrate analog Ap(CH2)pp to identify a single substrate binding site on the enzyme (19Dessauer C.W. Scully T.T. Gilman A.G. J. Biol. Chem. 1997; 272: 22272-22277Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Binding of Ap(CH2)pp to adenylyl cyclase is completely unaffected by the addition of a potent P-site inhibitor. Neither of the two cytosolic domains of adenylyl cyclase contains a classical nucleotide binding motif, although these domains share approximately 200 amino acid residues of similar sequence. These observations add credence to the possibility that the substrate binding and P sites on the enzyme are structurally distinct. The experiments described herein further define the nature of the P site and the mechanism of P-site inhibition. P-site inhibition is also used as a tool to facilitate understanding of the reaction catalyzed by adenylyl cyclases. 2′-[3H]Deoxyadenosine (7 Ci/mmol) was purchased from ICN and lyophilized regularly to remove [3H]H2O. Ap(CH2)pp was also purchased from ICN. Hexokinase and type XI glucose-6-phosphate dehydrogenase were purchased from Boehringer Mannheim and Sigma, respectively; these preparations were centrifuged, and the pelleted enzymes were resuspended in 20 mm NaHepes (pH 8.0) prior to use. Recombinant Gsα and the two cytosolic domains of adenylyl cyclase, VC1(591)Flag and IIC2, were purified after expression in Escherichia coli as described (16Whisnant R.E. Gilman A.G. Dessauer C.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6621-6625Crossref PubMed Scopus (117) Google Scholar, 18Sunahara R.K. Dessauer C.W. Whisnant R.E. Kleuss C. Gilman A.G. J. Biol. Chem. 1997; 272: 22265-22271Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 20Lee E. Linder M.E. Gilman A.G. Methods Enzymol. 1994; 237: 146-164Crossref PubMed Scopus (247) Google Scholar). Gsα was activated (whenever used) by incubation with 50 mm NaHepes (pH 8.0), 20 mm MgSO4, 1 mm EDTA, 2 mm dithiothreitol, and 400 μm GTPγS at 30 °C for 30 min; free GTPγS was then removed by gel filtration. Synthesis of cyclic AMP was measured as described (21Smigel M.D. J. Biol. Chem. 1986; 261: 1976-1982Abstract Full Text PDF PubMed Google Scholar) for 10–15 min at 30 °C in a final volume of 100 μl. GTPγS-Gsα was present (400 nm) unless otherwise indicated. Activities are expressed per mg of the limiting adenylyl cyclase domain in the assay (VC1). The other cytosolic domain (IIC2) was present in excess (1 μm) to drive the interaction between the two protein fragments. To determine kinetic constants, the concentration of MgATP was varied from 10 μm to 2.56 mm with a fixed excess of 10 mm Mg2+. Initial velocities were linear with time, and less than 10% of the ATP was consumed at the lowest substrate concentrations. All points were measured in duplicate and experiments shown were repeated two to four times. Values are reported ± S.E. of the mean. Synthesis of ATP from cyclic AMP and PPi, the reverse reaction, was measured spectrophotometrically in the presence of glucose, hexokinase, NADP, and glucose-6-phosphate dehydrogenase. Reaction velocities were calculated from the linear increase inA 340 resulting from the reduction of NADP. Reactions contained 20 mm NaHepes (pH 8.0), 50 mm glucose, 0.8 mm NADP, 3 mm free MgCl2, 1.7 units of hexokinase, and 0.3 units of glucose-6-phosphate dehydrogenase in a volume of 500 μl. The concentrations of substrates varied: 1.25–20 mm for cyclic AMP and 0.125–4 mm for MgPPi. PPiwas always added last to avoid precipitation. Reactions were typically started by addition of 0.4 μm VC1, 2 μm IIC2, and 1 μmGTPγS-Gsα (final concentrations) to the other reaction components (at 30 °C), and absorbance changes were measured for 10–15 min in a Beckman DU650 spectrophotometer with a temperature-controlled cuvette holder. The increase in absorbance in the absence of adenylyl cyclase was subtracted as background (<0.006 OD units/min). Optical densities of greater than 1.5 were excluded from analysis. Equilibrium dialysis was performed essentially as described (19Dessauer C.W. Scully T.T. Gilman A.G. J. Biol. Chem. 1997; 272: 22272-22277Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). To quantify binding of 2′-deoxyadenosine, each chamber contained 20 mm NaHepes (pH 8.0), 2.5 mm MgCl2, 2 mmdithiothreitol, 75 mm NaCl, 2′-[3H]deoxyadenosine (12.5–250 μm), and other additions as indicated. One chamber contained 18 μmVC1, 18 μm IIC2, and 25 μm GTPγS-Gsα; both of the cytosolic domains of adenylyl cyclase were necessary to observe binding. The opposite chamber contained buffer in lieu of the proteins. Samples were removed after dialysis for 24 h at 4 °C with rotation. Duplicate 15-μl aliquots from each chamber were analyzed by liquid scintillation spectrometry. Binding data have been normalized to protein concentrations based on the amount of active protein in the preparation, which was determined by titration with GTPγS-Gsα (19Dessauer C.W. Scully T.T. Gilman A.G. J. Biol. Chem. 1997; 272: 22272-22277Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Preparations of VC1 and IIC2 were more than 60% active by this criterion. We derived the rate equation for the reaction scheme presented in Fig. 5 using steady-state assumptions for the catalytic steps and rapid equilibrium for inhibitor binding. The critical features of the model include random release of products and no binding of P-site inhibitor to the free enzyme. The rates of both the forward and reverse reactions are complicated functions, described as v = [E t]*f([ATP], [cyclic AMP], [PPi], [2′-deoxyadenosine]), where v is the velocity in m/s and [E t] is the total molar concentration of enzyme. The equations that describe this function are presented in the "." Because of their complexity, they have not been rearranged to conform to standard Michaelis-Menten format. Curve fitting of individual experiments and modeling of kinetic data based on the steady-state rate equations were performed using Sigma Plot (Jandel Scientific). Inhibition of the Gsα-stimulated catalytic activity of our preparation of adenylyl cyclase by the P-site inhibitor 2′-deoxyadenosine is uncompetitive with respect to MgATP (Fig.1). These reactions (and those described below) were performed with a limiting concentration of the C1 domain of type V adenylyl cyclase and an excess of the C2 domain of type II adenylyl cyclase to drive the protein-protein interaction that is necessary for catalytic activity. Similar uncompetitive kinetics has been observed previously with a more potent P-site inhibitor, 2′-deoxy-3′-AMP, using Gsα-stimulated bovine brain adenylyl cyclase (22Johnson R.A. Shoshani I. J. Biol. Chem. 1990; 265: 11595-11600Abstract Full Text PDF PubMed Google Scholar) (presumably a mixture of isoforms) or a soluble system in which the two cytosolic domains of the enzyme were linked covalently (15Dessauer C.W. Gilman A.G. J. Biol. Chem. 1996; 271: 16967-16974Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Although 2′-deoxyadenosine has an unfortunately high K i (240 ± 60 μm) compared with 2′-deoxy-3′-AMP (5 μm, data not shown), we have utilized 2′-deoxyadenosine for this work because of the availability of the compound in radiolabeled form. Adenylyl cyclases (native enzymes or the soluble system utilized here) display noncompetitive or mixed noncompetitive inhibition by P-site analogs with respect to MnATP (3Londos C. Preston M.S. J. Biol. Chem. 1977; 252: 5957-5961Abstract Full Text PDF PubMed Google Scholar, 4Johnson R.A. Saur W. Jakobs K.H. J. Biol. Chem. 1979; 254: 1094-1101Abstract Full Text PDF PubMed Google Scholar, 10Wolff J. Londos C. Cook G.H. Arch. Biochem. Biophys. 1978; 191: 161-168Crossref PubMed Scopus (35) Google Scholar, 11Welton A.F. Simko B.A. Biochim. Biophys. Acta. 1980; 615: 252-261Crossref PubMed Scopus (8) Google Scholar, 15Dessauer C.W. Gilman A.G. J. Biol. Chem. 1996; 271: 16967-16974Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar,23Wong S.K.-F. Ross E.M. J. Biol. Chem. 1994; 269: 18968-18976Abstract Full Text PDF PubMed Google Scholar, 24Tang W.-J. Krupinski J. Gilman A.G. J. Biol. Chem. 1991; 266: 8595-8603Abstract Full Text PDF PubMed Google Scholar). We have focused our analysis on Gsα-activated adenylyl cyclase activity with MgATP as substrate (in the absence of Mn2+) to avoid the complications of mixed patterns of inhibition and the use of a second divalent cation (particularly Mn2+ in the presence of PPi, which has limited solubility). Uncompetitive inhibition implies that 2′-deoxyadenosine and ATP do not combine with the same form of the enzyme; noncompetitive inhibition is consistent with such a mechanism (but does not demand it). Florio (25.Florio, V. A. (1983) Inhibition of the Catalytic Unit of Mammalian Adenylate Cyclase by Adenosine and Adenosine Analogs.Ph.D. thesis dissertation, University of Virginia.Google Scholar) suggested that P-site ligands were dead-end inhibitors that formed a complex with the PPi-bound form of the enzyme (see Fig. 5). Alternatively, Johnson and Shoshani (22Johnson R.A. Shoshani I. J. Biol. Chem. 1990; 265: 11595-11600Abstract Full Text PDF PubMed Google Scholar) suggested that the P site is distinct from the active site and that both inhibitor and substrate could be bound simultaneously. To distinguish between these possibilities, we utilized equilibrium dialysis to examine directly the requirements for binding of 2′-deoxyadenosine to adenylyl cyclase. This was not possible previously because of limiting quantities of protein. We were unable to detect binding of 80 μm 2′-deoxyadenosine to 18 μm VC1 and IIC2 in the presence of Gsα and Mg2+ or Mn2+ (Fig.2 A); similar results were obtained after addition of the substrate analog Ap(CH2)pp. However, a modest level of binding was detected upon addition of 5 mm ATP under conditions (24-h incubation and very high enzyme concentration) where ATP and the products of the adenylyl cyclase reaction, cyclic AMP and PPi, should be in equilibrium. (The equilibrium constant, 0.065 m (26Hayaishi O. Greengard P. Colowick S.P. J. Biol. Chem. 1971; 246: 5840-5843Abstract Full Text PDF PubMed Google Scholar), implies final concentrations of roughly 0.33 mm ATP and 4.67 mm cyclic AMP and PPi.) We thus tested the capacity of cyclic AMP and PPi to support binding of 2′-deoxyadenosine. Binding of the P-site inhibitor was readily observed in the presence of PPi; this was not true in the case of cyclic AMP. Binding of 2′-deoxyadenosine required both VC1 and IIC2 and was not observed with the individual proteins in the presence or absence of Gsαand/or pyrophosphate (Fig. 2 A and data not shown). Analysis of 2′-[3H]deoxyadenosine binding to adenylyl cyclase in the presence of activated Gsα, Mg2+, and 2.5 mm PPi revealed a single binding site per C1/C2 heterodimer with a K d of 40 ± 3 μm (Fig.3). Both cyclic AMP and Ap(CH2)pp inhibited the binding of 2′-deoxyadenosine, implying that these molecules compete with P-site inhibitors for a single binding site (Fig. 2 B). Inhibition by the substrate analog, Ap(CH2)pp, renders highly unlikely the possibility of 2′-deoxyadenosine binding to a site that is distinct from the catalytic site but that still requires PPi to be manifest. This is the first direct evidence in support of Florio's hypothesis (25.Florio, V. A. (1983) Inhibition of the Catalytic Unit of Mammalian Adenylate Cyclase by Adenosine and Adenosine Analogs.Ph.D. thesis dissertation, University of Virginia.Google Scholar) of dead-end inhibition of adenylyl cyclase by P-site agents. Binding of a P-site inhibitor that is observable only in the presence of a reaction product suggests that release of product from the enzyme is at least partially rate-limiting. Patterns of inhibition of enzymatic activity by product are useful in determining if release of product is an ordered or random event. Adenylyl cyclase activity is inhibited by both reaction products, and previous studies with the enzyme from Brevibacterium liquefaciens demonstrated their random release (27.Wolin, M. S. (1981) Adenylate cyclase from Brevibacterium liquifaciens: A kinetic characterization of the catalytic mechanism and regulation of the enzyme by uncomplexed Mg2+ and pyruvate. Ph.D. thesis dissertation, Yale University.Google Scholar). Our enzyme system also displays random release of products, since the kinetics of inhibition of enzymatic activity by both cyclic AMP and PP is mixed; this is the pattern predicted by steady-state models. The intersection points for double-reciprocal plots (1/activity versus1/substrate concentration) at increasing concentrations of either product, PPi (Fig.4 A) or cyclic AMP (Fig.4 B), are above the abscissa and to the left of the ordinate. A purely competitive pattern of inhibition is expected for both products only if rapid equilibrium kinetics applies (28Segel I.H. Enzyme Kinetics. John Wiley & Sons, New York1975Google Scholar). The slopes of Fig. 4 B are not a linear function of cyclic AMP concentration and tend to curve upwards at high concentrations of the cyclic nucleotide. This is also indicative of a steady-state system where the assumption of rapid equilibrium does not apply (28Segel I.H. Enzyme Kinetics. John Wiley & Sons, New York1975Google Scholar). Finally, both cyclic AMP and PPi can compete with Ap(CH2)pp for binding to adenylyl cyclase (equilibrium dialysis data not shown); this again indicates that release of product is random. The equilibrium binding studies and patterns of product inhibition described to this point permit formulation of a model for inhibition of Gsα-stimulated adenylyl cyclase activity by P-site inhibitors (Fig. 5). We observe binding of 2′-deoxyadenosine only in the presence of PPi, and therefore the inhibitor is hypothesized to bind to only a single intermediate along the reaction coordinate. This is consistent with the uncompetitive kinetic pattern between P-site inhibitor and substrate. If P-site inhibitors bind to the enzyme in the absence of PPi, the interaction is apparently quite weak compared with that with the enzyme-PPi complex. Analysis of inhibition of adenylyl cyclase activity by both a P-site inhibitor and PPi or cyclic AMP provides additional kinetic information about the mechanism of inhibition and catalytically important steps. A diagnostic test for the interaction between two inhibitors is provided by Dixon plots of 1/velocity versusthe concentration of one inhibitor at a constant concentration of substrate and different fixed concentrations of the other inhibitor (28Segel I.H. Enzyme Kinetics. John Wiley & Sons, New York1975Google Scholar) (Fig. 6). The family of curves obtained at different 2′-deoxyadenosine concentrations with respect to cyclic AMP are parallel, indicating that the actions of these two inhibitors are mutually exclusive (Fig. 6 B). The IC50 for inhibition by cyclic AMP increases as the concentration of 2′-deoxyadenosine increases in a manner analogous to the interaction between a competitive inhibitor and substrate. This result is consistent with equilibrium binding data, which suggested a competitive interaction between cyclic AMP and 2′-deoxyadenosine. A Dixon plot of 1/velocity versus the concentration of PPi at different fixed concentrations of 2′-deoxyadenosine yields a family of intersecting lines, indicating that the two inhibitors do not bind to adenylyl cyclase in a mutually exclusive fashion (Fig. 6 A). The slope of the line for one inhibitor now depends on the concentration of the second. The intersection of these curves above the [PPi] axis indicates slight synergy between PPi and 2′-deoxyadenosine, and such synergy is anticipated from the binding data. However, the degree of synergy is dependent on a number of factors. The complexity of this result will be discussed below. The reaction catalyzed by adenylyl cyclase is readily reversible (26Hayaishi O. Greengard P. Colowick S.P. J. Biol. Chem. 1971; 246: 5840-5843Abstract Full Text PDF PubMed Google Scholar, 29Takai K. Kurashina Y. Suzuki C. Okamoto H. Ueki A. J. Biol. Chem. 1971; 246: 5843-5845Abstract Full Text PDF PubMed Google Scholar, 30Takai K. Kurashina Y. Hayaishi O. Methods Enzymol. 1974; 38: 160-169Crossref PubMed Scopus (2) Google Scholar). The equilibrium constant, measured with the enzyme from B. liquefaciens (26Hayaishi O. Greengard P. Colowick S.P. J. Biol. Chem. 1971; 246: 5840-5843Abstract Full Text PDF PubMed Google Scholar), is 0.065m (pH 7.3, 25 °C) and thus actually favors ATP synthesis under standard conditions of 1 m concentrations of reactants and products. The synthesis of ATP from cyclic AMP and PPi is activated by both Gsα and forskolin, as anticipated (Table I). The velocity of the reverse reaction is approximately 6% of that of the forward reaction assuming infinite substrate concentrations (TableII); a similar value was obtained with the pyruvate-stimulated bacterial enzyme (30Takai K. Kurashina Y. Hayaishi O. Methods Enzymol. 1974; 38: 160-169Crossref PubMed Scopus (2) Google Scholar).Table IForskolin- and Gsα-stimulation of ATP synthesis by adenylyl cyclaseNo activator1 μm Gsα50 μm forskolin1 μm Gsα + 50 μm forskolinActivity (μmol/min-mg)0.009 ± 0.0011.46 ± 0.0031.46 ± 0.073.43 ± 0.05Assays (0.3 μm VC1 and 2 μmIIC2) were performed in the presence of 2 mmPPi, 20 mm cyclic AMP, and the activator(s) indicated. Reactions performed in the absence of activator contained 2 μm VC1 and 10 μm IIC2. Synthesis of ATP was monitored by the increase in absorbance at 340 nm as described under Experimental Procedures. Open table in a new tab Table IIKinetic parameters: experimental data versus simulated fits to the steady-state rate equationForward reactionExperimental dataSimulated fitReverse reactionExperimental dataSimulated fitK m (ATP) (Figs. 1 and4)0.34 ± 90 mm0.26 mmK m (PPi)(Fig. 8 A)2-aValues of K m andV max for the reverse reaction are the apparent values obtained with the specified concentration of the second substrate and do not represent the K m or maximalV max of the system with infinite substrate concentrations. The calculated V max of the reverse reaction is 1.8 ± 0.1 s−1 versus 2.1 s−1 for the simulated fit.0.39 ± 0.09 mm0.37 mmmyentryV max (Figs. 1 and 4)30 ± 3 s−127 s−1V max(Fig. 8 A)2-aValues of K m andV max for the reverse reaction are the apparent values obtained with the specified concentration of the second substrate and do not represent the K m or maximalV max of the system with infinite substrate concentrations. The calculated V max of the reverse reaction is 1.8 ± 0.1 s−1 versus 2.1 s−1 for the simulated fit.1.0 ± 0.1 s−10.90 s−1myentryK i (app) 2′dAdo (Fig.1)240 ± 60 μm256 μmK m (cAMP) (Fig.8 B)2-aValues of K m andV max for the reverse reaction are the apparent values obtained with the specified concentration of the second substrate and do not represent the K m or maximalV max of the system with infinite substrate concentrations. The calculated V max of the reverse reaction is 1.8 ± 0.1 s−1 versus 2.1 s−1 for the simulated fit.8.7 ± 0.4 mm8.8 mmmyentryK i (PPi) (Fig.4 A)0.31 ± 0.02 mm0.16 mmV max (Fig.8 B)2-aValues of K m andV max for the reverse reaction are the apparent values obtained with the specified concentration of the second substrate and do not represent the K m or maximalV max of the system with infinite substrate concentrations. The calculated V max of the reverse reaction is 1.8 ± 0.1 s−1 versus 2.1 s−1 for the simulated fit.1.04 ± 0.05 s−11.33 s−1myentryK i (cAMP) (Fig.4 B)2.3 ± 0.4 mm2.7 mmK i (app) 2′dAdo (Fig.8 A)280 ± 30 μm284 μmmyentryK i (app)2′dAdo (Fig. 8 B)180 ± 30 μm160 μmmyentryK PPi(Fig. 7)0.12 ± 0.04 mm0.16 mmmyentryK cAMP (Fig. 7)2.3 ± 0.8 mm2.7 mmWe have fit data presented in Figs. 1, 4, 6, 7, and 8 to the steady-state rate equation shown in the "" to obtain estimates of K i and the rate constantsk 1 through k 12. These constants were used to plot simulations of the data, which are shown in Fig. 10, and these lines were used to calculate the values shown in the columns labeled "Simulated fit." The rate constants used to simulate experimental data are: k 1, 2.62 × 105m/s; k 2, 89.5 s−1;k 3, 59 s−1; k 4, 2.6 s−1; k 5, 0.8 s−1;k 6, 2.78 × 103m/s;k 7, 1060 s−1; k8, 1.11 × 105m/s; k 9, 0.39 s−1;k 10, 142 m/s; k 11, 56 s−1; k 12, 3.54 × 105m/s; K i, 150 μm. Experimental values for the forward and reverse reactions are reported as the average and the S.E. of the mean for two to four experiments. 2′dAdo, 2′-deoxyadenosine.2-a Values of K m andV max for the reverse reaction are the apparent values obtained with the specified concentration of the second substrate and do not represent the K m or maximalV max of the system with infinite substrate concentrations. The calculated V max of the reverse reaction is 1.8 ± 0.1 s−1 versus 2.1 s−1 for the simulated fit. Open table in a new tab Assays (0.3 μm VC1 and 2 μmIIC2) were performed in the presence of 2 mmPPi, 20 mm cyclic AMP, and the activator(s) indicated. Reactions performed in the absence of activator contained 2 μm VC1 and 10 μm IIC2. Synthesis of ATP was monitored by the increase in absorbance at 340 nm as described under Experimental Procedures. We have fit data presented in Figs. 1, 4, 6, 7, and 8 to the steady-state rate equation shown in the "" to obtain estimates of K i and the rate constantsk 1 through k 12. These constants were used to plot simulations of the data, which are shown in Fig. 10, and these lines were used to calculate the values shown in the columns labeled "Simulated fit." The rate constants used to simulate experimental data are: k 1, 2.62 × 105m/s; k 2, 89.5 s−1;k 3, 59 s−1; k 4, 2.6 s−1; k 5, 0.8 s−1;k 6, 2.78 × 103m/s;k 7, 1060 s−1; k8, 1.11 × 105m/s; k 9, 0.39 s−1;k 10, 142 m/s; k 11, 56 s−1; k 12, 3.54 × 105m/s; K i, 150 μm. Experimental values for the forward and reverse reactions are reported as the average and the S.E. of the mean for two to four experiments. 2′dAdo, 2′-deoxyadenosine. We have measured the rate of ATP synthesis at varying concentrations of the two substrates, cyclic AMP and PPi. The families of reciprocal plots (Figs. 7, Aand B) bo
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