P

presumably through a conformational change, inducing the dissociation of the P and Y subunits as the heterodimer GpY This stabilizes the resulting complex of Ga'GTP, which possesses the unique property of binding and activating adenylyl cyclase. So long as this complex persists, adenylyl cyclase is active and produces cAMP. However, the GTP is not perfectly stable in the complex of adenylyl cyclase and Ga-GTP, and it undergoes hydrolysis to GDP on a scale that is slow relative to enzymatic turnover. The complex of Ga-GDP does not retain the capacity to bind and activate adenylyl cyclase very well, presumably because of a conformation change induced by the hydrolysis of GTP, and the activation signal is switched off. Dissociation from adenylyl cyclase ensues, accompanied by re-binding of GpY to complete the cycle. These events take place on the surface of the membrane within which the hormone receptor and adenylyl cyclase remain anchored. The cycle allows the activity of adenylyl cyclase to be switched on so long as sufficient hormone is present. However, whenever the hormone is absent, the hydrolysis of GTP silences adenylyl cyclase. This brief discussion is limited to activation, but G proteins can also mediate inhibition of adenylyl cyclase in extended regulatory phenomena. The principle of G protein mediation extends to other signaling systems, including the ras P21 GTPase, which is implicated in cell proliferation and cancer.

The mechanism of action of adenylyl cyclase is under continuing investigation. Kinetic information indicates that cAMP dissociates from the product complex before MgPPi (Dessauer and Gilman, 1997). Membrane binding hampers progress on the reaction mechanism; however, the cytoplasmic domains can be expressed in E. coli. Soluble forms of activating G protein can also be expressed, and the three components undergo association to produce active adenylyl cyclase. Kinetic evidence implicates two divalent metal ions in the reaction mechanism (Garbers and Johnson, 1975). X-ray crystallographic analysis of complexes of the two cytoplasmic domains with Ga, ATP analogs, and Mg2+, Mn2+ or Zn2+ show the locations of two divalent metal ions at the active site (Tesmer et al., 1999). Shown in fig. 10-20 is the active site, with P-L-2',5'-dideoxy adenosine 3'-monophosphate bound with triphosphate and Mg2+. The two metal ions are linked together by mutual ligation to the conserved residues Asp396 and Asp440.

Consideration of the structure in fig. 10-20, together with other structures in which ATP aS is the ATP analog and Zn2+ is the divalent metal, leads to a proposal for the structure of a Michaelis complex between adenylyl cyclase and ATP (Tesmer et al., 1999). In this model, the 3'-OH group of ATP is ligated to MeA, lowering its pKa value and thereby increasing its nucleophilic reactivity for the internal displacement of pyrophosphate to form cAMP. Simultaneous coordination of MeA by Pa can be expected to increase the elec-trophilic reactivity of Pa toward nucleophilic attack by the 3'-oxygen. The mechanistic concept is outlined in fig. 10-21.

The postulated roles of the two metal ions in the mechanism of fig. 10-21 are similar to those proposed in the action of DNA polymerase. The biological roles of adenylyl cyclase and nucleic acid polymerases are different, but the underlying chemistry is similar. Moreover, the secondary structural motif of the PaPPaP fold defining the active site of adenylyl cyclase is similar to the palm-like domains in polymerases. Like the polymerases, a conforma-tional closing of the active site accompanies substrate binding (Tesmer et al., 1999).

Intervention in activation of adenylyl cyclase is an objective in pharmaceutical research. G protein mediated activation offers the possibility of blocking any of several steps in fig. 10-19 through the action of a drug. Compounds that inhibit adenylyl cyclase itself must directly block its action. The P-site inhibitors include naturally occurring compounds that bind to the active site, many with high affinity (Johnson et al., 1985; Dessauer and Gilman, 1996). These inhibitors are substrate analogs that cannot undergo cyclization. One of many such compounds is adenosine-2',5'-dideoxy-3'-triphosphate. This and other P-site

P-site

P-site

GTPyS

P-site

GTPyS

P-site

GTPyS

triphosphate

Lys1065

Arg1029

triphosphate

Lys1065

Arg1029

2, 5 -dideoxy adenosine 3 -monophosphate

Asp1018;

Lys938

triphosphate

2, 5 -dideoxy adenosine 3 -monophosphate

Asp1018;

Lys938

triphosphate

2, 5 -dideoxy adenosine 3 -monophosphate

Asp1018^ P-site

Lys938

P-site

2, 5 -dideoxy adenosine 3 -monophosphate

Asp1018^ P-site

Lys938

triphosphate °

Lys1065

Arg484

O"2

Arg1029

Asp396

Asp440

3 H2N

O Asn1025

2', 5'-dideoxyadenosine 3'-monophosphate

N17 N& Mjh

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