Kinetic characterization and ligand binding studies of His351 mutants of Bacillus anthracis adenylate cyclase.
Edema factor is a calmodulin dependent adenylyl cyclase secreted as one of the primary exotoxins by Bacillus anthracis. A histidine residue at position 351 located in its active site has been implicated in catalysis but direct evidence of its functional role is still lacking. In the present study, we introduced mutations in full-length edema factor (EF) to generate alanine (H351A), asparagine (H351N), and phenylalanine (H351F) variants. Spectral analysis of these variants displayed no gross structural deformities. Kinetic characterization showed that the adenylyl cyclase activity of H351N and H351F mutants decreased 34- and 40-fold, respectively, whereas H351A mutant completely lost activity. K(m) and K(i) values for ATP, pH activity profiles, and calmodulin activation curves of asparagine and phenylalanine mutants were not altered markedly. This kinetic data corroborated our ligand binding studies. Apparent K(d) values for calmodulin and ATP binding were found to be similar for wild-type EF and these active site variants. The effective substitution of H351 by asparagine and phenylalanine, albeit at a greatly reduced K(cat), without perturbing the ATP binding highlights the importance of this residue in transition-state stabilization. This was also evident from the positive free energy difference calculated for these mutants. However, equilibrium dialysis experiments revealed noticeable increase in ATP binding constant of H351A mutant, suggesting an additional role of H351 in precise substrate binding in the catalytic pocket. This is the first comprehensive study that describes the kinetic and ligand binding properties of H351 mutants and validates the importance of this residue in EF catalysis.
Gupta
Megha
M
Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India.
Alam
Sheeba
S
Bhatnagar
Rakesh
R
D000255:Adenosine Triphosphate / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000409:Alanine / Q000235:genetics; D000818:Animals; D001216:Asparagine / Q000235:genetics; D001408:Bacillus anthracis / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism; D002384:Catalysis; D006639:Histidine / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D010649:Phenylalanine / Q000235:genetics; D014757:Viper Venoms
D002147:Calmodulin; D014757:Viper Venoms; C416405:edema factor; D010649:Phenylalanine; D006639:Histidine; D001216:Asparagine; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases; D000409:Alanine