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′-[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.

• Publication year

  1997

• Venue

  Journal of Biological Chemistry

• Publication date

  1997-10-31

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1074/jbc.272.44.27787PMID 9346923
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• P-site analogs act as dead-end inhibitors of product release by stabilizing an E-PPi complex at the active site.

  Confidence 0.92

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewAK (4715169a40) review
• Product release from adenylyl cyclase is random, with cyclic AMP dissociating preferentially and pyrophosphate release being slow and partially rate-limiting.

  Confidence 0.95

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewAK (4715169a40) review
• 2′-deoxyadenosine binding at the P-site is observed only in the presence of pyrophosphate (PPi), and Ap(CH2)pp and cyclic AMP compete for that site under those conditions.

  Confidence 0.97

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewAK (4715169a40) review
• 2′-deoxyadenosine binds one site per C1/C2 heterodimer with a Kd of 40 ± 3 μM.

  Confidence 0.98

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewAK (4715169a40) review

• 2′-deoxyadenosine

  small molecule, ligand, inhibitor

  A radiolabeled deoxyadenosine ligand used to probe the P-site by equilibrium binding.

  Aliases: 2′-[3H]deoxyadenosine, 2'-deoxyadenosine

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewAK (4715169a40) review
• adenylyl cyclase

  enzyme

  The mammalian enzyme that catalyzes interconversion of ATP and cyclic AMP in the system studied.

  Aliases: AC

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewAK (4715169a40) review
• ap(ch2)pp

  substrate analog, small molecule

  A nonhydrolyzable adenosine triphosphate analog used as a substrate analog in competition experiments.

  Aliases: alpha,beta-methylene adenosine 5'-triphosphate, α,β-methylene adenosine 5′-triphosphate

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewAK (4715169a40) review
• c1/c2 heterodimer

  protein complex, enzyme complex

  The catalytic heterodimeric unit of mammalian adenylyl cyclase used as the binding stoichiometry reference.

  Aliases: C1/C2 dimer, C1/C2

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewAK (4715169a40) review
• cyclic amp

  metabolite, second messenger

  The cyclic nucleotide product of adenylyl cyclase that is examined for competition and dissociation behavior.

  Aliases: cAMP, cAMP cyclic AMP

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewAK (4715169a40) review
• e-ppi complex

  enzyme complex

  The enzyme-pyrophosphate complex used in the mechanistic model of adenylyl cyclase.

  Aliases: enzyme-product complex, E·PPi complex

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewAK (4715169a40) review
• product release

  reaction step, mechanistic step

  The step in which reaction products leave adenylyl cyclase after catalysis.

  Aliases: release of products

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewAK (4715169a40) review
• p-site

  binding site

  The inhibitor-binding site on adenylyl cyclase that accommodates P-site ligands and analogs.

  Aliases: P site

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewAK (4715169a40) review
• pyrophosphate (ppi)

  metabolite, product

  The pyrophosphate product associated with the enzyme-product state examined in the binding and kinetic experiments.

  Aliases: PPi, pyrophosphate

  박진우 (dztg5apj7m) extraction뀨 (7c402c1b98) reviewAK (4715169a40) review

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