Characterization of a beta-galactosidase hybrid protein carrying the catalytic domain of Escherichia coli adenylate cyclase.
A hybrid protein of Escherichia coli, exhibiting both adenylate cyclase and beta-galactosidase activities, was purified and characterized. This protein, obtained by genetic engineering, contained the first 556 amino acids of adenylate cyclase connected to the eighth-residue of beta-galactosidase through a pentapeptide Val-Gly-Asp-Pro-Val. The fusion protein was less stable than the native beta-galatosidase. Trypsin cleaved preferentially the adenylate cyclase moiety of the hybrid protein at a ratio of 1/50 (w/w). The kinetic properties of the hybrid protein were comparable, with a few exceptions, to those of native adenylate cyclase and beta-galactosidase. 'Truncated' adenylate cyclase was no longer sensitive to inhibition by excess ATP, which seems to indicate a second nucleotide binding site of wild-type adenylate cyclase. Photoirradiation of the hybrid protein with 8-azidoadenosine 5'-triphosphate inactivated the adenylate cyclase activity, leaving intact the beta-galactosidase activity. A radiolabeled ATP analog was incorporated after photoirradiation into the adenylate cyclase moiety of the fusion protein as shown by limited digestion with trypsin.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000345:Affinity Labels; D001386:Azides; D002384:Catalysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D005696:Galactosidases / Q000378:metabolism*; D007700:Kinetics; D010449:Peptide Mapping; D010777:Photochemistry; D014357:Trypsin; D001616:beta-Galactosidase / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D005696:Galactosidases; D001616:beta-Galactosidase; D014357:Trypsin; D000262:Adenylyl Cyclases