The Catalytic Mechanism of Mammalian Adenylyl Cyclase

The mechanism of P-site inhibition of adenylyl cyclase has been probed by equilibrium binding measurements using 2′-3Hdeoxyadenosine, 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′-3Hdeoxyadenosine, 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 of adenylyl we and have that the of the the structural for and adenylyl cyclase as as the features of P-site inhibition PubMed Scopus Google Scholar, J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, D. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We have utilized the substrate Ap(CH2)pp to a substrate binding site on the enzyme J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Binding of Ap(CH2)pp to adenylyl cyclase is by the of a P-site of the of adenylyl cyclase a binding these of These to the that the substrate binding and P sites on the enzyme are The the of the P site and the mechanism of P-site P-site inhibition is used as a to of the by adenylyl from and to Ap(CH2)pp from and were from and these were and the were in to and the of adenylyl and were in as Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). activated by with and at for by of cyclic AMP as J. Biol. Chem. Full Text PDF PubMed Google Scholar) for at in a of are per of the adenylyl cyclase in the assay The in to the interaction the protein kinetic the of from to with a of were with and of the ATP at the substrate were in and were to are ± of the of ATP from cyclic AMP and PPi, the reverse reaction, in the presence of and were from the from the of 3 of and of in a of The concentrations of for cyclic AMP and for to were typically by of and to the and were for in a with a The in in the absence of adenylyl cyclase as of were from as J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). binding of and as and both of the of adenylyl cyclase were to The in of the were for at with from were by Binding have been to protein concentrations based on the of active protein in the which by with J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of and were active by We the for the in using for the and equilibrium for The features of the model include release of products and no binding of P-site to the The of both the forward and reverse reactions are as is the in and is the of The that are in the of their have not been to to of and of kinetic based on the were using of the activity of of adenylyl cyclase by the P-site 2′-deoxyadenosine is uncompetitive with respect to These reactions those were with a of the of adenylyl cyclase and an of the of adenylyl cyclase to the interaction that is for uncompetitive has been observed with a P-site inhibitor, using adenylyl cyclase R.A. Shoshani I. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar) a of or a in which the of the enzyme were J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). Although 2′-deoxyadenosine has an K ± with 2′-deoxy-3′-AMP not we have utilized 2′-deoxyadenosine for of the of the in cyclases or the utilized noncompetitive or noncompetitive inhibition by P-site analogs with respect to (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, J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Ross E.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). We have analysis on adenylyl cyclase activity with as substrate (in the absence of to the of of inhibition and the of a divalent cation in the presence of PPi, which has inhibition that 2′-deoxyadenosine and ATP do not with the of the noncompetitive inhibition is with such a mechanism not A. of the of by Adenosine and Adenosine of Scholar) that P-site were dead-end inhibitors that a complex with the of the enzyme Johnson and Shoshani R.A. Shoshani I. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar) that the P site is from the active site and that both and substrate these we utilized equilibrium to the requirements for binding of 2′-deoxyadenosine to adenylyl not of of We were to binding of 2′-deoxyadenosine to and in the presence of and Mg2+ or were of the substrate a of binding of ATP and enzyme ATP and the products of the adenylyl cyclase reaction, cyclic AMP and PPi, in equilibrium J. Biol. Chem. Full Text PDF PubMed Google concentrations of ATP and cyclic AMP and We the of cyclic AMP and to support binding of Binding of the P-site observed in the presence of not in the of cyclic Binding of 2′-deoxyadenosine both and and not observed with the in the presence or absence of pyrophosphate A and not of binding to adenylyl cyclase in the presence of activated Gsα, and a binding site per C1/C2 with a K d of 40 ± 3 cyclic AMP and Ap(CH2)pp the binding of that these compete with P-site inhibitors for a binding site by the substrate analog, Ap(CH2)pp, the of 2′-deoxyadenosine binding to a site that is from the site but that to is the evidence in support of A. of the of by Adenosine and Adenosine of Scholar) of dead-end inhibition of adenylyl cyclase by P-site Binding of a P-site that is only in the presence of a product that release of product from the enzyme is at partially rate-limiting. of inhibition of activity by product are in release of product is an ordered or cyclase activity is by both and with the enzyme from their release M. S. cyclase from A kinetic of the mechanism and of the enzyme by Mg2+ and Scholar). enzyme release of the of inhibition of activity by both cyclic AMP and is is the by The for at concentrations of either or cyclic AMP are the and to the of the A of inhibition is for both products only equilibrium New Scholar). The of are not a of cyclic AMP and to at concentrations of the cyclic is of a the of equilibrium not New Scholar). both cyclic AMP and can compete with Ap(CH2)pp for binding to adenylyl cyclase not indicates that release of product is random. The equilibrium binding and of product inhibition to permit formulation of a model for inhibition of adenylyl cyclase activity by P-site inhibitors We binding of 2′-deoxyadenosine only in the presence of PPi, and the is to to only a the is with the uncompetitive kinetic P-site and P-site inhibitors to the enzyme in the absence of PPi, the interaction is with that with the of inhibition of adenylyl cyclase activity by both a P-site and or cyclic AMP kinetic the mechanism of inhibition and A for the interaction inhibitors is by of of one at a of substrate and concentrations of the New Scholar) The of at 2′-deoxyadenosine concentrations with respect to cyclic AMP are that the of these inhibitors are The for inhibition by cyclic AMP increases as the of 2′-deoxyadenosine increases in a to the interaction a and is with equilibrium binding which a interaction cyclic AMP and A of the of at concentrations of 2′-deoxyadenosine a of that the inhibitors do not to adenylyl cyclase in a The of the for one on the of the The of these the indicates and and such is from the binding the of is on a of The of The by adenylyl cyclase is J. Biol. Chem. Full Text PDF PubMed Google Scholar, C. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, 1974; 38: PubMed Scopus Google Scholar). The equilibrium with the enzyme from J. Biol. Chem. Full Text PDF PubMed Google is and ATP of concentrations of and The of ATP from cyclic AMP and is activated by both and as The of the reverse is of that of the forward substrate concentrations a with the enzyme 1974; 38: PubMed Scopus Google and of ATP by adenylyl ± ± ± ± and were in the presence of cyclic and the in the absence of and of ATP by the in at as in a to the ± of K for the reverse are the apparent with the of the substrate and do not the K or of the with substrate The of the reverse is ± for the ± and ± 3 of K for the reverse are the apparent with the of the substrate and do not the K or of the with substrate The of the reverse is ± for the ± ± of K for the reverse are the apparent with the of the substrate and do not the K or of the with substrate The of the reverse is ± for the ± ± of K for the reverse are the apparent with the of the substrate and do not the K or of the with substrate The of the reverse is ± for the ± ± ± ± ± ± have in and to the in the to of K and the These were used to of the which are in and these were used to the in the The used to are: K μm. for the forward and reverse reactions are as the and the of the for to of K for the reverse are the apparent with the of the substrate and do not the K or of the with substrate The of the reverse is ± for the in a and were in the presence of cyclic and the in the absence of and of ATP by the in at as We have in and to the in the to of K and the These were used to of the which are in and these were used to the in the The used to are: K μm. for the forward and reverse reactions are as the and the of the for to We have the of ATP at concentrations of the cyclic AMP and The of both at a the and These that the binding of one substrate the of the The for the binding of to enzyme can from of the or New Scholar). The K d for binding of cyclic AMP to adenylyl K is ± while that for PPi, is ± of the apparent or provide the of the at substrate concentrations ± ± and the apparent for binding of the substrate ± 3 ± and to binding but for cyclic AMP and PPi, New Scholar). analysis of the of concentrations of 2′-deoxyadenosine on the of the reverse reaction, at concentrations of PPi, of uncompetitive 2′-deoxyadenosine and with of the or with P-site A. of the of by Adenosine and Adenosine of Scholar) dead-end model of P-site inhibition a interaction 2′-deoxyadenosine and cyclic AMP with to the of the reverse is in concentrations of the the of for which cyclic AMP and P-site inhibitors The apparent K for is ± with d of 40 by equilibrium for binding of the to adenylyl cyclase in the presence of concentrations of a K for at concentrations of of an K of not The d and the K to the in the for these measurements at at of equilibrium to at in of analysis of product we the for the model in We have in the of the for the of Mg2+ as a in the and the in the of by of of the in and permit of of the kinetic A of the K and those by the of is in We do not that the in but do the kinetic observed we the of the The only at which the from the are in the of in of 2′-deoxyadenosine and the of inhibition by cyclic AMP and is the that are and in the forward the release of cyclic AMP to the release of from the in the reverse the enzyme to cyclic the of the forward appear to 3 and the of of ATP and the release of PPi, These the of P-site inhibition of their of the of the We have the for a of for the various inhibition to the as the model in as by of to the and analysis of an ordered product release in which the release of cyclic AMP the release of PPi, to inhibition by cyclic AMP in the forward and that permit binding of P-site analogs to both the and the enzyme do not uncompetitive inhibition by 2′-deoxyadenosine when the of the P-site for the complex that for the enzyme by of in increases the of both the forward and reverse reactions and the apparent K for 2′-deoxyadenosine the of inhibition by the P-site The K d for binding of 2′-deoxyadenosine to adenylyl cyclase in the presence of is with the observed in the presence of ± of adenylyl cyclase by the of and the for and adenylyl ± ± ± ± ± ± and the for 2′-deoxyadenosine with respect to cyclic AMP ± and the for 2′-deoxyadenosine with respect to cyclic AMP ± and the apparent K for 2′-deoxyadenosine with respect to ± and the apparent K for 2′-deoxyadenosine with respect to of the forward and and reverse and reactions were as in the to and with the of include the of the from and the for 2′-deoxyadenosine with respect to cyclic AMP and the apparent K for 2′-deoxyadenosine with respect to in a of the forward and and reverse and reactions were as in the to and with the of include the of the from cyclase a ATP binding site that at the the and of the protein J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). P-site inhibitors have no on the binding of the substrate Ap(CH2)pp to site J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is in the uncompetitive of P-site inhibition with respect to A uncompetitive not to the the to the enzyme only substrate is mechanisms for P-site inhibition have been The of a dead-end by M. S. cyclase from A kinetic of the mechanism and of the enzyme by Mg2+ and Scholar) to inhibition of adenylyl cyclase from by that uncompetitive inhibition by adenosine the of an complex at the site that not in equilibrium with active of the A. of the of by Adenosine and Adenosine of Scholar) and and Ross V.A. Ross E.M. Mol. Pharmacol. 1983; 24: 195-202PubMed Google Scholar) a mechanism for adenylyl a dead-end complex PPi, and P-site mechanism by Johnson and Shoshani R.A. Shoshani I. J. Biol. Chem. 1990; Full Text PDF PubMed Google in which P-site and substrate to sites on the of an mechanism V.A. Ross E.M. Mol. Pharmacol. 1983; 24: 195-202PubMed Google of the of the a binding site. the in P-site we have used equilibrium to requirements for binding of were not with enzyme in the presence of Ap(CH2)pp or cyclic binding in the presence of the product one of 2′-deoxyadenosine per C1/C2 with a K d of 40 ± 3 μm. Cyclic AMP or Ap(CH2)pp or These provide evidence to support the that P-site analogs act as dead-end inhibitors of product by and of both and adenylyl cyclases indicates that the release of product is R.A. Shoshani I. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, M. S. cyclase from A kinetic of the mechanism and of the enzyme by Mg2+ and Scholar). and the binding permit formulation of the in kinetic support mechanism and provide product the inhibition of the forward by both 2′-deoxyadenosine and products or for cyclic AMP or PPi, that cyclic AMP and P-site inhibitors act in a the of the is with the that both to the site. The that the interaction 2′-deoxyadenosine and the indicates that both are to the enzyme and as by with adenylyl cyclase activity in and the for dead-end A. of the of by Adenosine and Adenosine of Scholar). have observed a interaction 2′-deoxyadenosine and with not Johnson and Shoshani to any these inhibitors using a adenylyl cyclase activated with R.A. Shoshani I. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). their model of an using the for dead-end inhibition that of can depending on the The and for ATP, PPi, and cyclic AMP to the of observed with the of 2′-deoxyadenosine and as inhibitors of the forward in by in adenylyl cyclase or of to of apparent P-site inhibitors and of ATP by adenylyl cyclase a substantial of the binding of cyclic PPi, and of the reverse by 2′-deoxyadenosine is uncompetitive with respect to PPi, that binding of interaction of with the by 2′-deoxyadenosine is with respect to cyclic AMP in the presence of concentrations of PPi, which the of both cyclic AMP and 2′-deoxyadenosine compete for the binding and kinetic for the reverse that a the of PPi, and the is with cyclic AMP at the site of the The model in is not only with the kinetic but with features of P-site These inhibitors a strict for an (2Londos C. Wolff J. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5482-5486Crossref PubMed Scopus (489) Google J. Londos C. Cooper D.M.F. Adv. Cyclic Nucleotide Res. 1981; 14: 199-214PubMed Google Scholar, R.A. Yeung S.-M.H. Stübner D. Bushfield M. Shoshani I. Mol. Pharmacol. 1989; 35: 681-688PubMed Google with binding at the site. and evidence from that the active site at the of the and J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google PubMed Scopus Google Scholar). A that the for P-site inhibition in adenylyl is at PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar). P-site inhibitors a have a potency (12Johnson R.A. Yeung S.-M.H. Stübner D. Bushfield M. Shoshani I. Mol. Pharmacol. 1989; 35: 681-688PubMed Google and that the complex a to a both and a a Furthermore, the site of several and at the are P-site and at are (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). of adenylyl cyclases to various adenosine analogs in the of their active sites to the of these analogs R.A. L. Shoshani I. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of adenylyl cyclase are to P-site inhibition are of the enzyme (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). is by a dead-end P-site inhibition of the forward reaction, release of the enzyme at partially the complex at sufficient concentrations to that in model are of the enzyme that to the release of such that product release is no the potency of P-site is the of that a of a P-site to the is in the of of cyclic AMP and is slow to the of release of these not and P-site inhibitors of adenylyl cyclase by or the of of cyclic AMP and to concentrations of and potency of P-site the complex a and the of the the of adenylyl cyclase for P-site Binding and kinetic for and adenylyl cyclase with the that both mechanisms to the potency of P-site of to adenylyl cyclase a in of the forward and reverse The apparent K for 2′-deoxyadenosine is as d for binding of 2′-deoxyadenosine is in the presence of both that the complex a the binding that and the activity of adenylyl cyclase by stabilizing the of the A of P-site inhibition is the potency of these when analogs of ATP are used as R.A. Shoshani I. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). These analogs have either a for the enzyme or the of the release of product is no with the of the reaction, and the potency of P-site inhibitors The of P-site as a of M. PubMed Scopus Google Scholar) can by any in in K d for such that the to release of The inhibition of the forward by P-site inhibitors is noncompetitive or noncompetitive with respect to (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 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, J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the enzyme that are with binding and permit binding to the P-site analogs can inhibit the enzyme in the presence of both in the presence and absence of ATP Johnson R.A. J. Biol. Chem. 1990; Full Text PDF PubMed Google with the of binding of these to both and the enzyme these several of evidence P-site inhibitors are not of with a substrate for binding to the enzyme in the presence of and at concentrations the K for inhibition J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we have not observed binding to adenylyl cyclase in the presence of absence of with the that 2′-deoxyadenosine is a inhibitor, and we not to binding the is in the of the K d for a substrate analog, Ap(CH2)pp, by with Mg2+ J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Binding with inhibitors structural in the and P-site We for for in the and and Ross for The is by and is the of the the of enzyme and the are by and and are as 3 The complex designated and are of are in with the in