Escherichia coli adenylate cyclase at

Chapter V: The catalytic and regulatory domain of Escherichia coli adenylate cyclase

Escherichia coli adenylate cyclase is a monomeric protein of 848 amino acids and molecular weight 97,586.

The existence of a catalytic domain was foreseen by Wang JY, 1981 in an elegant article titled "Molecular cloning and amplification of the adenylate cyclase gene".  It was found 'intriguing' that a 67,000 dalton peptide had some activity, although it had less activity than an 80,000 dalton one.  Since then, many truncated adenylate cyclases have been characterized that retained some activity.  In the 1981 article, it was justly argued that an essential activator must be present to obtain full adenylate cyclase activity, and 'perhaps' it was the product of the crr gene that encodes Enzyme IIAGlc.

Roy A, 1983 proposed adenylate cyclase was composed of two functional domains, an amino-terminal catalytic domain and a carboxy-terminal regulatory domain.  Such proposal was related to the observation that the carboxy-terminal domain was required for the glucose-mediated regulation of adenylate cyclase.  The existence of two functional domains was further emphasized upon availability of the adenylate cyclase gene full sequence (Aiba H, 1984).  In addition, the regulatory domain was required for the CRP-dependent activation of adenylate cyclase by phosphorylated Enzyme IIAGlc(Crasnier M, 1990)

The catalytic domain of Escherichia coli adenylate cyclase was further characterized as a tryptic fragment of molecular weight 30,000, with an amino- and carboxy-terminus corresponding respectively to residue 82 and 341 of adenylate cyclase (Holland MM, 1988).  Surprisingly, the 30,000 dalton peptide had kinetic properties similar to those reported for full-length adenylate cyclase.  Presently, this unexpected finding may be understood by postulating adenylate cyclase activity is heavily dependent on the activation by phosphorylated Enzyme IIAGlc.

In 1994, a genetic approach provided the characterization of a 48,000 dalton amino-terminal domain that synthesized in vivo 10 times more cAMP than wild type adenylate cyclase.  Cyclic AMP synthesis by this 'over-active' domain was observed in strains lacking Enzyme IIAGlc (Crasnier M, 1994).  Consequently, a molecular model of interaction between domains was proposed describing the regulatory carboxy-terminal domain as inhibitory to the activity of the catalytic domain.  Thus, it was tentatively suggested that phosphorylated Enzyme IIAGlc activates adenylate cyclase by relieving inhibitory interactions between domains.  Unfortunately, in vitro data are not presently available demonstrating a direct interaction between the regulatory domain of adenylate cyclase and phosphorylated Enzyme IIAGlc.

In 2005, it was considered unlikely that the carboxy-terminal domain of adenylate cyclase is (i) necessary for the glucose-mediated regulation of adenylate cylase and (ii) inhibitory to the catalytic domain (Strozen TG, 2005).  However, experimental data supporting such conclusions were scarce, and the two adenylate cyclase mutants analyzed (C-terminal truncated adenylate cyclase of presumably 507 and 631 amino acids) were not characterized, especially by cAMP measurements.  These mutants were reported to be less active than wild type adenylate cyclase.  Because the rescuing effect observed with these mutants could not be reproduced in wild type strains grown on glucose, the conclusion, the carboxy-terminal domain of adenylate cyclase is not regulatory, is not experimentally supported.  In 2006, the proposal that the carboxy-terminal domain is regulatory upon interaction with phosphorylated Enzyme IIAGlc was reiterated (Park YH, 2006).

The simplistic view, adenylate cyclase is composed of two functional domains, does not reflect the complexity of the molecular interactions occurring within its likely multiple structural domains.  It does, however, reflect most of the experimental data obtained so far in vivo.

Chapter VI