Properties of F1-ATPase from the uncD412 mutant of Escherichia coli

Abstract

Properties of purified F1-ATPase from Escherichia coli mutant strain AN484 (uncD412) have been studied in an attempt to understand why the amino acid substitution in the beta-subunit of this enzyme causes a tenfold reduction from normal MgATP hydrolysis rate. In most properties that were studied, uncD412 F1-ATPase resembled normal E. coli F1-ATPase. Both enzymes were found to contain a total of six adenine-nucleotide-binding sites, of which three were found to be non-exchangeable and three were exchangeable (catalytic) sites. Binding of the non-hydrolysable substrate analogue adenosine 5'-[beta gamma-imido]triphosphate (p[NH]ppA) to the three exchangeable sites showed apparent negative co-operativity. The binding affinities for p[NH]ppA, and also ADP, at the exchangeable sites were similar in the two enzymes. Both enzymes were inhibited by efrapeptin, aurovertin and p[NH]ppA, and were inactivated by dicyclohexylcarbodi-imide, 4-chloro-7-nitrobenzofurazan and p-fluorosulphonyl-benzoyl-5'-adenosine. Km values for CaATP and MgATP were similar in the two enzymes. uncD412 F1-ATPase was abnormally unstable at high pH, and dissociated into subunits readily with consequent loss of activity. The reason for the impairment of catalysis in uncD412 F1-ATPase cannot be stated with certainty from these studies. However we discuss the possibility that the mutation interrupts subunit interaction, thereby causing a partial impairment in the site-site co-operativity which is required for 'promotion' of catalysis in this enzyme.

Structure of the nucleotide‐binding domain in the β‐subunit of Escherichia coli F1‐ATPase

“…The binding change mechanism (see [26] for reviews) requires that conformational changes at alternate catalytic sites be tightly coupled to each other. The alternating arrangement of the cy-and &subunits in Fr [27,28] and the results of the uncA @-subunit) mutant studies of Wise et al [29,30] indicate that this involves a pathway of conformational signal transmission from the nucleotide-binding domain of one&subunit through a neighbouring a-subunit to an alternate P-subunit [1,31]. A possible clue to the pathway of the conformational signal transmission within the &subunit comes from examination of the location in fig.2 of residues which, when altered, disrupt the conformational coupling (positive catalytic cooperativity) between Fr-ATPase catalytic sites.…”

Photoaffinity cross‐linking of F1ATPase from spinach chloroplasts by 3'‐arylazido‐β‐alanyl‐8‐azido ATP

“…The number of nucleotide binding sites have been in debate for a long time. Based on results for photoaffinity labeling of CF, by 2-azidoadenine nucleotides three catalytic and three noncatalytic nucleotide binding sites are now believed to be present per CF1 [5] in analogy to the F,ATPases from mitochondria [6] and bacteria [7], but smaller numbers have been discussed too [8,9]. The position of these nucleotide binding sites could not be cleared finally.…”

The Subunit b Dimer of the FoF1-ATP Synthase

“…Routine Analysis-Protein concentrations were determined according to the method of Bradford (56) using defatted bovine serum albumin (Roche Applied Science) as standard. ATP hydrolysis was assayed at 30°C and pH 8.0 with 2-3 g of enzyme as described by Wise et al (59). The production of inorganic phosphate was detected by the method of Taussky and Shorr (60).…”