Adenylyl cyclase is a lyase class enzyme that catalyzes the formation of cyclic adenosine monophosphate from adenosine triphosphate. Adenylyl cyclase exists in three classes (I, II, and III) and has nine isoforms in class III mammalian adenylyl cyclase. It exists mainly in a membrane associated form but can also be found in a soluble form depending on the organism or tissue. Adenylyl cyclase is a major player in maintaining signal transduction pathways by converting numerous extracellular hormonal signals into the form of a second messenger in cyclic adenosine monophosphate. The active site, as well as some regulatory sites, is formed as a result of dimerization of the enzymes two catalytic domains, which is essential for catalysis. Regulation of adenylyl cyclase is performed by activated G-proteins, calmodulin, P-site analog inhibitors, phosphorylation, and forskolin, a hypotensive diterpene drug, at differing regulatory sites. Two Mg2+ or Mn2+ ions are required to bind with adenosine triphosphate in order for catalysis to occur. The production and regulation of cyclic adenosine monophosphate is critically important in the activation of several cellular metabolic processes, including enzyme activity, gene-expression patterns, and membrane excitability, making correct adenylyl cyclase function essential in the maintenance of normal physiology.
 

Introduction:
 Adenylyl cyclase (AC), EC 4.6.1.1., is a lyase class enzyme that catalyzes the conversion of ATP to 3',5'-cAMP (cyclic adenosine monophosphate) and pyrophosphate (Figure 1) (1,2,3,4). AC is

also known by other common names such as adenylate cyclase and 3',5'-cAMP synthetase (5). AC exists in most cells as a transmembrane protein in small amounts less than 10 pmols/mg of membrane, but is also found in soluble form in the cytoplasm of some cell types and unicellular organisms (2,6,7). However, mammalian transmembrane AC and soluble AC are suspected to have evolved separately based upon the greater similarities of portions of the catalytic domains of sAC with those of bacterial AC than with transmembrane AC (8). AC acts as a signal converter as it receives signals from hormones and other molecules that ultimately activate it to catalyze the formation of 3',5'-cAMP, an important second messenger in signal transduction pathways (9). The signal that AC helps to transduce comes from various external hormonal stimuli that bind to and activate membrane associated G-proteins. 
 Most forms of AC are regulated by G-proteins. Upon receiving hormonal stimulus at the receptor complex, a conformational change occurs in the cytoplasmic heterotrimeric G-protein. Of the G-protein subunits Ga, GB, and Gy, Ga is of most importance as it is activated by this conformational change by a decrease in affinity for the already bound GDP and an increase in affinity for GTP. Binding of GTP denotes a release of Ga from GBy. G? then is considered to be one of two forms, Gia or Gsa. These two forms of Ga allosterically regulate AC’s catalytic activity, inhibiting AC activity in the case of Gia and stimulating AC activity in the case of Gsa. Stimulation of AC activity leads to increased intracellular concentration of 3',5'-cAMP, and inhibition decreases 3',5'-cAMP production (Figs. 1 and 2) (9,10,11). 3',5'-cAMP then relays the signal to the interior of the cell where many cellular metabolic processes are effected (12).

Structure:
 There are three classes of AC, however, the structure of the active site is highly conserved across all classes. Class I-ACs are found in Gram negative bacteria such as E coli. Class II-ACs, or “toxic” AC, are found in pathogenic microbes such as Bordetella pertussis and Bacillus anthracis. Finally, class III-ACs are found in a wide range of organisms, including nine isoforms, AC-1 through AC-9, found in mammals (7,13). AC has cytoplasmic soluble (sAC) forms and membrane associated forms. The membrane associated forms consist of two transmembrane domains of 6 a-helices each. Both the soluble and membrane associated forms have two highly conserved cytoplasm associated catalytic domains, C1 and C2. In the membrane associated forms, C1 bridges the two transmembrane domains and C2 makes up the C-terminal end of the protein. C1 and C2 come together to form separate active and regulatory sites at their interface (9,14). 
Regions of C1 and C2, namely C1a and C2a, have been shown through mutational analysis to contribute the amino acids that make up the ATP binding and active site. The active site is comprised primarily of structures a1, B1, the B2-B3 turn of the C1a domain, and  4' and  5' of the C2a domain (10). The regulatory site at the interface of the joined catalytic domains is a potential binding site for ATP, but, in most mammalian isoforms, has a greater affinity for forskolin, a hypotensive diterpene usually administered to patients with hypothyroidism. Forskolin binds at a large hydrophobic pocket that lies proximal to the active site, thus, stabilizing the C1/ C2 dimer (10,15). Another, more significant, allosteric site lies approximately 30 Å from the catalytic site. This site also forms as a result of the C1/ C2 dimerization and is the site of Gsa binding. Thus, Gsa, along with forskolin, stabilize the C1/ C2 dimer, activating AC. Gsa is the GTP activated stimulatory alpha subunit of a heterotrimeric G-protein after it receives appropriate hormonal stimulus (7,10). The Gsa site is 1800 Ų and includes a region that is complementary to the switch 2 region of Gsa, which is important in inducing an activating conformational change of AC (Fig. 3) (10). The GBy G-protein dimeric subunit upregulates AC-I and II at sites other than the Gs? allosteric site on C2. Noncompetive inhibition of AC is accomplished by binding of Gia inhibitory subunit of Gi family of G-proteins to a currently unidentified regulatory site (7,10).
 Ca2+, in the form of calmodulin, stimulates the activity of AC-I and AC-VIII and inhibits activity of AC-III and AC-IX. Ca2+ binds to and activates calmodulin, however, the mechanism and site at which calmodulin regulates these forms of AC is unknown. Ca2+ has also been shown to inhibit AC-V and AC-VI, with the entry of external Ca2+ concentrations having a greater affect on inhibition than the release of internal stores of Ca2+. Also phosphorylation by protein kinase A (PKA) or PKC of Ser674 in the C1b region reduces AC-VI activity by 50%. An analog of ATP, P-site inhibitor, shows a higher affinity within the active site of AC and has been used extensively to determine the kinetics and mechanism of AC (10).

Kinetics and Catalytic Mechanism:
AC has a turnover number range of 1-100 s-1 and a Km value range of 30-400 microM. AC, at least the mammalian forms, exhibits random sequential displacement with a slight preference for cAMP being release first from the active site. AC exists in three conformations corresponding to the unactivated form (E), the substrate-free activated form (E*), and the substrate/product bound form (E**) (Fig. 4). Upon binding of activators such as Gsa, forskolin, and the P-site analog, the E state is suspected to undergo a conformational change at the C1/C2 interface to form the E* state, which includes the formation of C1/C2 dimerization. It is also thought that further conformational change occurs upon binding of substrate to allow for catalysis. For correct binding and catalysis, cofactors, such as Mg2+ in transmembrane AC and Mn2+ in soluble AC, are required in pairs (10). 
 In order for AC to catalyze the conversion of ATP into 3',5'-cAMP, ATP must bind so that the 3'-OH group of the ribose closely approaches the a-phosphorus. The 3'-OH group becomes activated by base catalysis for attack on the a-phosphorus, forming the 3',5',a-phosphorus ring within cAMP and displacing pyrophosphate (Fig. 1). 

 Within the active site, residues Asp354, Arg398, Lys938, Arg1011, Asp1018, Asn1025, Arg1029,  and Lys1067 are involved in ATP-Mg2+/Mn2+ binding and catalysis (3,10). These residues are brought into correct position by a 7° rotation of the C1/C2 domains with respect to each other during activator induced dimerization. The adenine ring of ATP binds to a hydrophobic pocket within the active site and is also stabilized by water mediated interaction between the polar amine group and Asn1025. However, the greatest specificity for adenine is determined by hydrogen bonding between the Asp1018 and the adenine N6, as well as hydrogen bonding of Lys938 and the adenine N1. These two residues, Asp1018 and Lys938, differentiate AC from being a GTP binding guanyl cyclase (GC) (10,16). 
 To stabilize the remainder of the substrate, Lys1067 binds with one of the negatively charged phosphate groups. Lys1067 is part of a carboxyl terminal “lid” region (residues 1058-1071) that folds down over the substrate after the initial binding of ATP-Mg2+/Mn2+. The phosphate groups of ATP are also stabilized by two Mg2+/Mn2+ and by Arg398 and Arg1011, which are contributed by C1 and C2, respectively. The two Mg2+/Mn2+ ions are themselves stabilized in position for catalysis by two aspartate residues of currently unknown position, one contributed from each catalytic domain as well. However, only one of the two ions is suspected to be directly involved in catalytic action (10,16). 
 Based on mutagenesis and kinetic analysis, Asn1025, Arg1029, and Asp354 are the residues most directly involved with catalysis. Arg1029 is thought to be located close to the a-phosphorus and is thought to interact  with the bridging oxygens of the a-phosphate. Asn1025 is also thought to interact with the bridging oxygens and assist Arg1029 in stabilizing the transition state or leaving group. Asp354 is thought to act as a base in deprotonating the 3'-OH group that then attacks the a-phosphorus by nucleophilic attack. This nucleophilic attack by the 3' oxyanion releases pyrophosphate from ATP and forms 3',5'-cAMP (10,16).
 

Diseases: 
 AC acts as a signal transducer within signal transduction from the exterior of a cell to the interior, so any AC mutation or compound that alters normal AC function and/or regulation will either cut the transduction pathway short or indefinitely perpetuate the pathway. Also, since AC produces the second messenger cAMP, the normal cellular metabolic functions, such as enzyme activity, gene-expression patterns, and membrane excitability, regulated by cAMP will be altered. cAMP regulates a wide variety of cellular functions by activating forms of protein kinase (Fig. 5). 

The functions affected depend upon the hormonal stimulus and the hormone’s target tissue. For example, when the hormone adrenaline binds to G-protein associated receptors in cells of the heart, cAMP is upregulated by the activation of AC. cAMP then stimulates an increase in heart rate and contraction strength. The same hormonal stimulus targets muscle and fat cells, but causes different responses. In muscle, cAMP induces glycogen breakdown, and in fat cells, cAMP induces lypolysis. Thus, the abnormal production or regulation of cAMP could have major detrimental effects on these metabolic processes. G-proteins are regulated by numerous other hormones including glucagon, secretin, and parathyroid, among others, which all confer induction of separate signal transduction cascades. However, all theses cascades are sustained by only a few forms of AC within an organism, making AC’s function vitally important in cellular metabolic function (7,9,10,17). This makes implications of disease and metabolic abnormalities associated with AC dysfunction due to mutations or disease almost endless.  Examples of disease caused by AC dysfunction or dysfunction in AC regulation include whooping cough and malignant hyperthermia. Whooping cough is caused by the pertussis toxin produced by the bacterium Bordetella pertussis. Ultimately pertussis toxin blocks inhibition of AC by Gia leading to an overproduction of cAMP in multiple tissues. This overproduction disrupts normal functions regulated by cAMP, conferring the diseased state, in this case, whooping cough (17). Malignant hyperthermia (MH) is an example of the effects of high AC activity. MH results from a high cAMP concentration in skeletal muscle that accumulates as a result of abnormally elevated AC activity (18).
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