Pertussis Active site

Figure 6.3. Mechanism of action of heterotrimeric G-proteins. Upon receptor occupancy, the Ga-subunit binds GTP in exchange for GDP, and then moves in the membrane until it encounters its target enzyme, shown here as adenylate cyclase (alternatively, a phospholipase). The activated target enzyme then becomes functional. Inherent GTPase activity within the a-subunit then hydrolyses bound GTP to GDP, and the a-subunit dissociates from its target enzyme (which becomes inactive) and rebinds the / - and ysubunits. Upon continued receptor occupancy, further catalytic cycles of GTP exchange and target enzyme activation may occur. The scheme shown is for a stimulatory G-protein (Got,), but similar sequences of events occur with inhibitory G-proteins (Gcx,) except that the interaction of the a-subunit with adenylate cyclase will result in its inhibition. The sites of action of pertussis and cholera toxins are shown.

Got subunits of the Gi/o type of G proteins can be ADP-ribosylated in the presence of pertussis toxin at Cys351, four amino acids from the C-terminus. Petussis toxin sensitivity is the major method of identifying a role for Gai/o proteins in GPCR-mediated signaling. This treatment prevents receptor-mediated G-protein activation and thus exchange of GTP for GDP and so blocks signaling by Ga and G(3y. There are numerous examples of the use of this technique to identify coupling of the delta opioid receptor [e.g., 2,3,41,42,73,77]. One Ga protein in this class, Gaz, lacks the Cys residue that is the site for pertussis toxin action and so is insensitive to pertussis toxin treatment [see 17 for review]. 

Lobet Y, Feron C, Dequesne G, et al. (1993) Site-specific alterations in the B-oligomer that affect receptor-binding activities and mitogenicity of pertussis toxin. In J. Exp. Med. 177 79—87. 

Locht C, Lobet Y, Feron C, et al. (1990) The role of cysteine 41 in the enzymatic activities of the pertussis toxin SI subunit as investigated by site-directed mutagenesis. In J. Biol. Chem. 265 4552-4559. 

PertOfran desipramine. pertussis toxin (PTX) is elaborated by a bacterium Bordetella pertussis) and is a hexameric protein (4-5 subunits from A-B complex). It is a G-protein inactivator that binds to the ADP-ribosylation regulatory site of the G/Go family of subunits which couple negatively to adenylyl cyclase. The cellular responses blocked by PTX are varied, and typically include those due to 03 and opioid receptor type activation. The inactivation of this key regulatory unit explains some of the side-effects of whooping cough (caused by Bordetella pertussis) where production of this toxin is a main pathological factor. This toxin is an important pharmacological tool. 

Cholera and pertussis toxins catalyze the covalent addition of ADP-ribose to specific sites in the ot subunits of Gs and Gi, respectively. This modification inhibits GTPase action in the ot subunits and converts them to irreversible activators of adenylate cyclase. As a result, cAMP accumulates. In the intestine, the response to this is an uncontrollable secretion of water and sodium—causing severe diarrhea and dehydration. 

The synthesis of the two diastereoisomers of P -l-(2-nitrophenyl)ethyl adenosine S -lri-phosphate (91) has been achieved using resolved (R)- and (5)-l-(2-nilroidienyl)ethanol. The alcohols were converted to (R)- and (5)-l-(2-nitrophenyl)ethyl phosphates by phosphitylation with N,)V-diisopropyl-fi(s-(2-cyanoethyl)phosphoramidite (92) and subsequent oxidation with 3-chlorobenzoic acid. Each of the monophosphates was activated with carbonyidiimidazole and condensed with adenosine diphosphate to give the desired triphosphate. These ATP analogues can be used for the rapid release (by flash photolysis) of ATP in biological systems. The 8-azido-3 -0-anthraniloyl derivatives of 2 -dADP (93) and 2 -dATP (94) have been prepared in seven steps from 8-azido-2 -deoxyadenosine. These compounds are of interest as fluorescent and photoactivatable probes for the nucleotide binding site of kinases and cyclases. In particular, (94) was shown to be a competitive inhibitor of Bordetella pertussis adenylate cyclase and the observed K- (74 pM) was close to tiiat predicted from the K- value of 3 -0-anthraniloyl-2 -dATP. ... 

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