The role of histidine 63 in the catalytic mechanism of Bordetella pertussis adenylate cyclase.
Of the 9 histidines located in the catalytic domain of Bordetella pertussis adenylate cyclase, three (His63, His106, and His298) were found to be conserved in the adenylate cyclase of Bacillus anthracis, another calmodulin-dependent enzyme. Substitution of His63 with Arg, Glu, Gln, or Val decreased the catalytic efficiency of adenylate cyclase between 2 and 3 orders of magnitude and altered the kinetic properties of the enzyme. These effects varied in relation to the nature of the substituting residue, pH, and direction of the reaction, i.e. ATP cyclization (forward) or ATP synthesis (reverse). Arg was the best substituent for His63 as catalyst in the forward reaction, with shift of the optimum pH to the alkaline side, whereas Glu was the best substituent for His63 in the reverse reaction, with shift of the optimum pH to the acidic side. Diethyl pyrocarbonate, which had a deleterious effect on wild-type adenylate cyclase was ineffective on His63 mutants. From these results we conclude that His63 is involved in the reaction mechanism of adenylate cyclase, which requires a general acid/base catalyst, most probably as an intermediate in a charge-relay system.
Munier
H
H
Unité de Biochimie des Régulations Cellulaires, Institut Pasteur, Paris, France.
Bouhss
A
A
Krin
E
E
Danchin
A
A
Gilles
A M
AM
Glaser
P
P
Bârzu
O
O
D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001886:Bordetella pertussis / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003062:Codon; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000378:metabolism
D003062:Codon; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006639:Histidine; D000262:Adenylyl Cyclases; D004047:Diethyl Pyrocarbonate