Purification and characterization of the inactive Ca 2+, Mg 2+-activated adenosine triphosphatase of the unc A − mutant Escherichia coli AN120

Abstract

The inactive Ca 2+, Mg 2+-activated ATPase of the une A − mutant Escherichia coli AN120 and the active enzyme of the normal strain AN180 were purified to homogeneity. Both enzymes contained five different subunits (α-ϵ) with the same molecular weights and stoichiometry. Both enzymes gave identical cross-linked products with the bifunctional reagent dithiobis(succinimidyl propionate), suggesting that the subunits were arranged in the same way in the two enzymes. Treatment of the enzyme of AN120 with N atosylphenylalanine chloromethyl ketone-trypsin did not reveal latent ATPase activity. The ATPase of AN120 bound normally to ATPase-depleted membranes of AN180 or AN120 to reconstitute aerobic-driven energy-dependent transhydrogenase activity. The enzymes from both AN120 and AN180 contained one to two molecules of both ATP and ADP per molecule of ATPase. Each enzyme could also bind one molecule of exogenous [ 3H]ADP per molecule of ATPase. The dissociation constants of the [ 3H]ADP-ATPase complexes formed by the enzymes from AN120 and AN180 were 3.55 and 9.3 μ m, respectively. Both enzymes reacted with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole with the substitution of a tyrosine residue at the active site. It is concluded that the loss of hydrolytic activity in the ATPase of AN120 is due to an alteration at the active site which is reflected in nucleotide binding but may involve a catalytically active amino acid.