Site of Adenylylation

The P-site of adenylyl cyclase inhibits cyclic AMP accumulation 308 There are four adenosine receptor subtypes 308 Xanthines block P2 but not P2 receptors 309... 

The P-site of adenylyl cyclase inhibits cyclic AMP accumulation. Since P, and P2 receptors are located on the cell surface, they bind purines or pyrimidines in the extracellular space. There also is an adenosine binding site located intracellularly on the enzyme adenylyl cyclase (see Ch. 21). This is referred to as the P-site of adenylyl cyclase. Binding of adenosine and other purines, notably 3 AMP, 2 deoxy-3 -ATP and 2, 5 -dideoxyadenosine to this site, inhibits adenylyl cyclase activity [8]. The P-site of adenylyl cyclase and other intracellular purine binding sites are not classified as purinergic receptors. 

Davies and coworkers found that streptomycin adenylyl transferase also adenylylates spectinomycin and actinamine. Because of the stereochemical resemblance between actinamine and the o threo methyl-amino alcohol moiety in streptomycin, the site of adenylylation was considered to be that shown by the arrow in formula 15. 

The production of preparative amounts of homogeneously adenylylated Rabl allows detailed binding studies of the modified protein with regulatory factors (that are GEF, GAP, GDI) and downstream effectors. As the site of adenylylation is situated in the highly important switch II region, this modification would hypothetically interfere with the binding of most proteins. The facile production of the modified protein ensures that even interactions of moderate-to-weak affinity can be quantified. 

Adenylyl Cyclases. Figure 4 Regulation of adenylyl cyclases by G-proteins. Abbreviations Hs, Hj, Rs, and Rj denote hormones and receptors that lead to stimulation or inhibition, respectively, of adenylyl cyclases, Ca and Ci are active and inactive configurations of adenylyl cyclase, Fo forskolin binding site, Gs and Gj are GTP-dependent regulatory proteins comprising their respective as, and (3y subunits. 

An early observation that 2 -d-3 -AMP was a more potent inhibitor of adenylyl cyclases than 2 -d-Ado suggested that the enzyme would accept substitutions at the 3 -ribose position and that phosphate was particularly well tolerated. This led to the generation of a family of 3 -phosphoryl derivatives of 2, 5 -dideoxyadenosine exhibiting ever greater inhibition with the addition of an increasing number of 3 -phosphoryl groups, the most potent of which is 2, 5 -dideoxyadenosine-3 -tetraphosphate (2, 5 -dd-3 -A4P Table 4) [5]. These constitute a class of inhibitors historically referred to as P -site ligands that caused inhibition of adenylyl... 

Probably all adenylyl cyclases are inhibited competitively by substrate analogs, which bind at the site and to the enzyme configuration with which cation-ATP binds (cf Fig. 4). One of the best competitive inhibitors is (3-L-2, 3 -dideoxy adenosine-5 -triphosphate ( 3-L-2, 3 -dd-5 -ATP Table 4) [4], which allowed the identification of the two metal sites within the catalytic active site (cf Fig. 4) [3]. This ligand has also been labeled with 32P in the (3-phosphate and is a useful ligand for reversible, binding displacement assays of adenylyl cyclases [4]. The two inhibitors, 2, 5 -dd-3 -ATP and 3-L-2, 3 -dd-5 -ATP, are comparably potent... 

P-site ligands inhibit adenylyl cyclases by a noncompetitive, dead-end- (post-transition-state) mechanism (cf. Fig. 6). Typically this is observed when reactions are conducted with Mn2+ or Mg2+ on forskolin- or hormone-activated adenylyl cyclases. However, under- some circumstances, uncompetitive inhibition has been noted. This is typically observed with enzyme that has been stably activated with GTPyS, with Mg2+ as cation. That this is the mechanism of P-site inhibition was most clearly demonstrated with expressed chimeric adenylyl cyclase studied by the reverse reaction. Under these conditions, inhibition by 2 -d-3 -AMP was competitive with cAMP. That is, the P-site is not a site per se, but rather an enzyme configuration and these ligands bind to the post-transition-state configuration from which product has left, but before the enzyme cycles to accept new substrate. Consequently, as post-transition-state inhibitors, P-site ligands are remarkably potent and specific inhibitors of adenylyl cyclases and have been used in many studies of tissue and cell function to suppress cAMP formation. 

There are also many neurotransmitter and hormone receptors that contribute to the fine control of cAMP formation by inhibition of adenylyl cyclase. The action of inhibitory receptors is mediated by several different forms of the Gai family, specifically the Gail, Gai2, Gai3, Gao and Goa subtypes. The Ga subunits of these isoforms can inhibit the catalytic activity of adenylyl cyclase when the enzyme is activated by either Gas or forskolin. The inhibition of catalytic activity does not occur via competition with Gas but appears to occur by an interaction at a symmetric site on the AC molecule. Gai-mediated inhibition of adenylyl cyclase is most dramatic for AC5 and AC6. A few other forms of adenylyl cyclase, most notably AC1, can be inhibited by Gao but this effect is not as potent as the inhibition of AC5 and AC6 by Gai isoforms. The GTPase activity of Gai family members can be accelerated by a large family of RGS proteins (see Chapter 19). 

The activation of adenylyl cyclase enables it to catalyze the conversion of adenosine triphosphate (ATP) to 3 5 -cyclic adenosine monophosphate (cAMP), which in turn can activate a number of enzymes known as kinases. Each kinase phosphorylates a specific protein or proteins. Such phosphorylation reactions are known to be involved in the opening of some calcium channels as well as in the activation of other enzymes. In this system, the receptor is in the membrane with its binding site on the outer surface. The G protein is totally within the membrane while the adenylyl cyclase is within the membrane but projects into the interior of the cell. The cAMP is generated within the cell (see Rgure 10.4). 

A further consequence of the conformational change in the a-subunit induced by the Y-phosphate is the activation of the effector molecule next in sequence. The binding site of the sequential effector molecule adenylyl cyclase includes the switch II (Tes-mer et al., 1997). It is therefore assumed that the conformational change of switch II also mediates the binding and activation of the effector molecule. The binding site for the effector and for the Pycomplex partially overlap, so that a binding of the effector is only possible if the Py-complex has dissociated. 

Fig. 5.22. Topology of adenylyl cyclase. The adenylyl cyclase of mammals is a transmembrane protein. It is composed of two homologons domains, which each have a transmembrane domain (Ml and M2) and a larger cytoplasmic portion (Cl and C2). Sequence analysis predicts 6 transmembrane hehces for each of the domains (numbering from 1-12). The active site is formed by residues from Cl and C2.

Regulation of protein kinase A by cAMP takes place by the following mechanism. An increase in cAMP concentration, triggered by activation of adenylyl cyclase, leads to binding of cAMP at specific binding sites on the regulatory subunit. The R subunits dissociate from the tetramer, the catalytic subunits are released from inhibition by the regulatory subunits and can thus phosphorylate substrate proteins. 

Muscarinic M2 Myocardium, smooth muscle, some presynaptic sites CNS neurons Opening of potassium channels, inhibition of adenylyl cyclase... 

When the nucleotide-binding site of Gs (on the a subunit) is occupied by GTP, Gs is active and can activate adenylyl cyclase (AC in Fig. 12-12) with GDP bound to the site, Gs is inactive. Binding of epinephrine enables the receptor to catalyze displacement of bound GDP by GTP, converting Gs to its active form (step (2)). As this occurs, the /3 and y subunits of Gs dissociate from the a subunit, and Gsa, with its bound GTP, moves in the plane of the membrane from the receptor to a nearby molecule of adenylyl cyclase (step (3)). The Gsa is held to the membrane by a covalently attached palmitoyl group (see Fig. 11-14). 

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