Adenylate cyclase, stereochemical

Hewlett AC, Champion TM, Wilken GH, Mechoulam R, Stereochemical effects of 11-OH-A -tetrahydrocannabinol-dimethylheptyl to inhibit adenylate cyclase and bind to the cannabinoid receptor. Neuropharmacology 29 161—165, 1990. 

Burstein, S. H. et al., Detection of Cannabinoid Receptors by Photoaffinity Labelling," Biochem. Biophys. Res. Commun., 176(l) 492-497 (Apr. 15,1991). Hewlett, A. C., et al., Stereochemical Effects of ll-OH-A8-lfetrahydrocan-nabinol-Dimethylheptyl to Inhibit Adenylate Cyclase and Bind to the Cannabinoid Receptor," Neuropharmacology, 29(2) 161-165 (Feb. 1990). 

The first application of this 0 effect for determining the configuration of an oxygen chiral phosphate ester was the author s determination of the configuration of diastereomeric samples of cyclic [, 0]dAMP, the chiral substrate for studying the stereochemical consequences of the reverse reaction catalyzed by adenylate cyclase (formation of cyclic AMP from ATP), and of the hydrolysis reaction catalyzed by 3, 5 -cyclic nucleotide phosphodiesterase (25) (see Fig. 

A conceptually similar approach can be used to determine the configurations of chiral phosphate monoesters of chiral alcohols without the necessity for transferring the chiral phosphoryl group to 1,3-butanediol. For example, in a number of stereochemical studies of hydrolysis reactions performed in the author s laboratory, 3 - or 5 -nucleotides were obtained as reaction products. In the case of samples of 5 -AMP, these can be enzymically converted to isotopically labeled cyclic 3, 5 -nucleotides by initial pyrophosphorylation with adenylate kinase and pyruvate kinase, followed by enzymic cyclization with inversion of configuration by a bacterial adenylate cyclase (20) (see Figs. 6 and 7). In the case of samples of 3 -nucleotides or 5 -nucleotides other than 5 -AMP, the cyclization reactions can be accomplished chemically (23). In either case, following chemical con-... 

These comparative studies constituted the first example of an enzyme-catalyzed hydrolysis reaction whose stereochemical course was unaffected by sulfur substitution. At the time these experiments were performed, the stereochemical courses of the reactions catalyzed by glycerol kinase (83, 84) and by the bacterial adenylate cyclase (85, 86) had already been compared in the laboratories of Knowles and Gerlt, respectively, and these were also found to be unaffected by the sulfur substitution. A number of other comparisons of this type have been made, and in no case were the stereochemical consequences of the reactions studied with chiral phosphate esters and the chiral thiophosphate analogs found to differ. This agreement suggests that the necessary use of oxygen chiral thiophosphate monoesters to study the stereochemical course of phospho-monoesterases will provide pertinent results for ascertaining whether phosphory-lated intermediates are involved in the reaction mechanism. 

Nucleotidyl cyclases catalyse the formation of 3, 5 cyclic nucleotide monophosphates from nucleotide triphosphates. Adenylate cyclase, which catalyses the formation of cAMP, is of particular importance because of its major biological role in regulating the level of cAMP and hence regulating cellular communication. Study of the mechanism of enzyme-catalysed cycli-zation has been hindered through the lack of pure enzyme preparations— adenylate cyclases are membrane-bound proteins. However, Coderre and Gerlt have completed stereochemical analyses of the accessible adenylate cyclase from Brevibacterium liquefaciens. 

Few details are available about the chemical mechanisms of the reactions catalyzed by nucleotidyl cyclases since these enzymes can be associated with membranes and therefore difficult to purify and/or are present in very small amounts and therefore difficult to obtain in amounts compatible with many modem enzymological techniques. We have decided to direct our attention to bacterial adenylate cyclases because one, the enzyme produced by Brevi-bacterium liquefaciens, has been purified to homogeneity (Takai et al., 1974) and the gene for another, that from Salmonella typhimurium. has been cloned in a multiple-copy plasmid (Wang et ai, 1981). Given the limited amounts of enzymes that are presently available, chemical studies designed to ascertain directly if the reaction occurs via formation and breakdown of an adenylated enzyme intermediate are impossible therefore, we decided to use a stereochemical approach to obtain information regarding the existence of an adenylated enzyme intermediate in the reaction catalyzed by the cyclase isolated from B. liquefaciens. 

Hayaishi and colleagues, who devised the purification for the Brevibacter-ium liquefaciens enzyme, used it to characterize the reversibility of the adenylate cyclase reaction (Kurashina et ai, 1974) and found that the equilibrium constant for the reaction written in the direction of cyclic AMP formation is 0.12 Mat pH 7.3 at this pH the rates of the forward and reverse reactions are comparable but about the rate of the forward reaction measured at its pH optimum, pH 9. Our plan for determining the stereochemical course of the reaction is shown in Fig. 14. Since we had synthesized the diastereomers of cyclic [, 0]dAMP, we would use the cyclase to catalyze their pyrophosphorolysis and form the diastereomers of [a- 0, 0]dATP. However, the thermodynamics of the cyclase reaction prevents an efficient conversion of cyclic dAMP to dATP, so this reaction was coupled to the glycerol kinase reaction the kinase reaction utilizes the thermodynamic instability of the )J,y-anhydride bond to displace the overall equilibrium to favor the synthesis of the diastereomers of [a- 0, 0]dADP. Both the cyclase and glycerol kinase can utilize deoxyadenosine nucleotides as substrates, but only the cyclase reaction can alter the configuration of the chiral phosphorus atoms. 

Fig. 14. Strategy for determining the stereochemical course of the reaction catalyzed by the adenylate cyclase from Brevibacterium liquefaciens. From Codene and Gerlt (1980). Copyright 1980 American Chemical Society.

This result is very persuasive evidence that the mechanism of the adenylate cyclase reaction does not involve the formation of an adenylated enzyme intermediate. In addition, this study provided the second demonstration that oxygen chiral and phosphorothioate substrates are processed by enzymes with the same stereochemical course we had previously found that the Sp diastereomer of ATPaS is converted to the Rp diastereomer of cyclic AMPS with this cyclase (Gerlt et ai, 1980a). The first example of sulfur not altering the stereochemical course of an enzymatic reaction was provided by... 

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