Functional groups at the catalytic site of BF1 adenosinetriphosphatase from Escherichia coli.

The rates of inactivation of BF1 adenosinetriphosphatase (BF1-ATPase) from Escherichia coli by 7-chloro-4-nitro-2,1,3-benzoxadiazole, 1-fluoro-2,4-dinitrobenzene, 2,4,6-trinitrobenzenesulfonate, phenylglyoxal, and N,N'-dicyclohexylcarbodiimide have been measured in the presence and absence of various concentrations of inorganic phosphate, ADP, ATP, or magnesium ion. Dissociation equilibrium constants and rate constants for the labeling reactions have been deduced from a quantitative treatment of the kinetic data. The results suggest that the essential Tyr, Lys, Arg, and Glu or Asp residues are probably located at the catalytic site of BF1-ATPase and that in addition to steric interference, the effect of charge interaction should also be considered in interpreting the kinetic data on the protection of BF1-ATPase by substrate molecules against inactivation by the above labeling reagents. Examination of the relative values of the rate constants for the labeling reactions in the presence and absence of inorganic phosphate, ADP, ATP, or magnesium ion, respectively, and the effect of NBD label on the rates of labeling of the essential guanidinium, amino, and carboxyl groups suggest that the arrangement of these four functional groups at the catalytic site of BF1 may be similar to that previously proposed for MF1-ATPase from bovine heart; namely, the essential amino group and the unusually reactive phenol group are probably located near the bound inorganic phosphate or the gamma-phosphate group of the bound ATP, the essential guanidinium group is probably located nearer to the alpha- or beta-phosphate group than to the gamma-phosphate group of the bound ATP or the bound inorganic phosphate, and the essential carboxylate group is probably complexed with a magnesium ion which it shares with the bound inorganic phosphate.