J. Biol. Chem., Vol. 258, Issue 6, 3750-3758, 03, 1983

Purification and characterization of adenylate cyclase from Escherichia coli K12

JK Yang and W Epstein

Adenylate cyclase of Escherichia coli K12 has been purified 17,000-fold to near homogeneity from a 5-fold overproducing strain. One major band of Mr = 92,000 and several minor bands are seen on sodium dodecyl sulfate-polyacrylamide electrophoresis of the purest fractions. Identification of the enzyme with the 92,000-Da protein is based on the correlation of this band with activity when highly purified enzyme is eluted from ADP-sepharose columns. The native enzyme has a molecular weight of 95,000 determined by gel filtration, showing that the enzyme is active as a monomer. The purest enzyme has a specific activity of 700 nmol min-1 mg-1, indicating a turnover number of about 100 min-1. Our data indicate that there are only about 15 molecules of the enzyme in wild type cells of E. coli. In crude extracts, over 80% of the activity is soluble after centrifugation at 100,000 x g, indicating the enzyme is soluble or, at most, loosely membrane bound. The enzyme is only moderately stable in crude extracts and becomes more unstable as purification proceeds. Activity is stabilized by ATP, or at -20 degrees C as an ammonium sulfate precipitate or in 50% glycerol. The enzyme has an absolute requirement for divalent cations. Maximum activity with Mg2+ is reached at 30 mM. Mn2+ is a good substitute; Co2+ activates well at low concentrations but becomes inhibitory at high concentrations; and Ca2+ is a potent inhibitor in the presence of Mg2+. The isoelectric point of the enzyme is 6.1, and its pH optimum is 8.5. The enzyme is inhibited by its substrate, with a Km of about 1 mM and a Ki of about 1.5 mM, and is noncompetitively inhibited by PPi, ADP, GTP, and a number of other compounds. The data suggest that dissociation of PPi from the first enzyme-product complex is the rate-limiting step in the reaction. Activation of the enzyme, inferred to occur in vivo, could be produced by a postulated regulatory effector which speeds release of PPi from the enzyme-product complex.

This article has been cited by other articles:

				K. Bettenbrock, T. Sauter, K. Jahreis, A. Kremling, J. W. Lengeler, and E.-D. Gilles Correlation between Growth Rates, EIIACrr Phosphorylation, and Intracellular Cyclic AMP Levels in Escherichia coli K-12 J. Bacteriol., October 1, 2007; 189(19): 6891 - 6900. [Abstract] [Full Text] [PDF]

				J. Deutscher, C. Francke, and P. W. Postma How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria Microbiol. Mol. Biol. Rev., December 1, 2006; 70(4): 939 - 1031. [Abstract] [Full Text] [PDF]

				Y.-H. Park, B. R. Lee, Y.-J. Seok, and A. Peterkofsky In Vitro Reconstitution of Catabolite Repression in Escherichia coli J. Biol. Chem., March 10, 2006; 281(10): 6448 - 6454. [Abstract] [Full Text] [PDF]

				J. Tellez-Sosa, N. Soberon, A. Vega-Segura, M. E. Torres-Marquez, and M. A. Cevallos The Rhizobium etli cyaC Product: Characterization of a Novel Adenylate Cyclase Class J. Bacteriol., July 1, 2002; 184(13): 3560 - 3568. [Abstract] [Full Text] [PDF]

				T. Eppler, P. Postma, A. Schutz, U. Volker, and W. Boos Glycerol-3-Phosphate-Induced Catabolite Repression in Escherichia coli J. Bacteriol., June 1, 2002; 184(11): 3044 - 3052. [Abstract] [Full Text] [PDF]

				E. Krin, O. Sismeiro, A. Danchin, and P. N. Bertin The regulation of Enzyme IIAGlc expression controls adenylate cyclase activity in Escherichia coli Microbiology, May 1, 2002; 148(5): 1553 - 1559. [Abstract] [Full Text] [PDF]

				H.-J. Kim, W.-T. Chang, M. Meima, J. D. Gross, and P. Schaap A Novel Adenylyl Cyclase Detected in Rapidly Developing Mutants of Dictyostelium J. Biol. Chem., November 20, 1998; 273(47): 30859 - 30862. [Abstract] [Full Text] [PDF]

				O. Sismeiro, P. Trotot, F. Biville, C. Vivares, and A. Danchin Aeromonas hydrophila Adenylyl Cyclase 2: a New Class of Adenylyl Cyclases with Thermophilic Properties and Sequence Similarities to Proteins from Hyperthermophilic Archaebacteria J. Bacteriol., July 1, 1998; 180(13): 3339 - 3344. [Abstract] [Full Text]

				J.-i. Kawabe, Y. Toya, C. Schwencke, N. Oka, T. Ebina, and Y. Ishikawa Soluble Adenylyl Cyclase from Spodoptera frugiperda (Sf9) Cells. PURIFICATION AND BIOCHEMICAL CHARACTERIZATION J. Biol. Chem., August 16, 1996; 271(33): 20132 - 20137. [Abstract] [Full Text] [PDF]