In 1983, the purification and subsequent in vitro characterization of the Escherichia coli adenylate cyclase protein was reported by Yang and Epstein [J Biol Chem]. The authors established that adenylate cyclase is a monomer of molecular weight 95,000 (as determined by gel filtration), in good agreement with the calculated molecular weight of 97,586.
The protein was also reported to be soluble (possibly loosely membrane bound), and unstable during the purification process, a finding in agreement with an instability index that classifies adenylate cyclase as unstable (the instability index can be obtained by using the ProtParam tool from ExPASy, the Expert Protein Analysis System). The protein could be stabilized upon addition of ATP.
The isoelectric point was found to be 6.1, a result in agreement with the theoretical one of 5.81. Under standard assay conditions, activity was maximal at pH 8.5 and falling off rapidly above and below the optimum pH (for reference E. coli intracellular pH is slightly basic).
Other particular features of adenylate cyclase as reported by Yang and Epstein are summarized below.
The enzymatic activity is quite low, with a turnover number of about 100 per minute. This is in contrast with in vivo rates of cAMP synthesis that are at least an order of magnitude higher (when measured in conditions leading to maximal activity). The enzyme is ionic strength sensitive, requires a high divalent cation concentration and is more active in a less polar environment. Finally, a kinetic analysis indicated that pyrophosphate (PPi) is the first product released, with subsequent formation of a cAMP-enzyme complex. The release of PPi is slow and rate-limiting. Consequently, the authors proposed that a regulator (possibly the product of the crr gene encoding Enzyme IIAGlc) may increase the rate of PPi dissociation.
Among other properties of adenylate cyclase worth mentioning, Tao and Huberman reported a total inhibition of its activity in the presence of mercuric acetate. This inhibition was relieved in the presence of dithiothreitol [PubMed]. Sulfhydryl groups may therefore be essential to adenylate cyclase activity (there are 15 cysteine residues in adenylate cyclase or 1.77% of total amino-acids).
In 1983, the purification of Escherichia coli adenylate cyclase by Yang and Epstein was a major accomplishment. Since that time in vitro studies of purified adenylate cyclase have, unfortunately, been rare. In particular the putative regulation by phosphorylated Enzyme IIAGlc was never established in vitro. This is somewhat puzzling considering the constant interest for cAMP and its major role in E. coli and affiliated bacteria.