Escherichia coli adenylate cyclase at

Chapter VII: Purification of adenylate cyclase

The purification, and subsequent in vitro characterization of Escherichia coli adenylate cyclase, was reported by Yang JK, 1983.  The authors established adenylate cyclase was a monomer of molecular weight 95,000 as determined by gel filtration, in agreement with the molecular weight of 97,586 which was calculated later on.

The protein was 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 however 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 JK, 1983 are summarized as follows.  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 being slow and rate-limiting.  Thus 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, a total inhibition of activity in the presence of mercuric acetate was reported.  This inhibition was relieved in the presence of dithiothreitol (Tao M, 1970).  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).

The purification of E. coli adenylate cyclase by Yang JK, 1983 was a major accomplishment.  Since that time, in vitro studies of purified adenylate cyclase have unfortunately been inconclusive.  In particular, the putative activation by phosphorylated Enzyme IIAGlc has not been established in vitro.  This is somewhat puzzling considering the constant interest for cAMP, and its major physiological role in E. coli and affiliated bacteria.