Mutations in the conserved proline 43 residue of the uncE protein (subunit c) of Escherichia coli F1F0-ATPase alter the coupling of F1 to F0 (original) (raw)
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Escherichia coli ATP synthase (F-ATPase): catalytic site and regulation of H+ translocation
The Journal of experimental biology, 1992
We discuss our recent results on the Escherichia coli F-ATPase, in particular its catalytic site in the beta subunit and regulation of H+ transport by the gamma subunit. Affinity labelling experiments suggest that beta Lys-155 in the glycine-rich sequence is near the gamma-phosphate moiety of ATP bound at the catalytic site. The enzyme loses activity upon introduction of missense mutations in beta Lys-155 or beta Thr-156 and changes catalytic properties upon introduction of other mutations. By analysis of mutations and their pseudo revertants, residues beta Ser-174, beta Glu-192 and beta Val-198 were found to be located near the glycine-rich sequence. The combined approaches of chemical labelling and genetics have been fruitful in visualizing the structure of the catalytic site. Analysis of mutations in the gamma subunit suggests that this subunit has an essential role in coupling catalysis with proton translocation.
Archives of Biochemistry and Biophysics, 1983
Various hybrid plasmids carrying a portion of the gene for the y subunit of the H'-ATPase of Escherichia coli complemented five mutants defective in the enzyme in a genetic test, indicating that the mutants are defective in the y subunit. Since the nucleotide sequence of genomic DNA carried on the plasmids is known, the defective site(s) of the mutants could be located within the gene for the y subunit as follows: KFlO and NR70, KFl, and KF12 and KF13 have a mutation causing a defect(s) in amino acid residues 1 to 82, 83 to 167, and 168 to 287, respectively, of the y subunit. The biochemical properties of all these mutants except NR70 were analyzed in terms of proton permeability of the membranes and assembly of F1. Results suggested that KFl and KFlO have defective F1 without at least the cy and p subunits on their membranes, whereas KF12 and KF13 have F1's of rather similar structure to that of the wild type. Attempts were made to purify F1 of KF12 as a single complex. Although the F1 complex dissociated during purification, active CY and /3 subunits of KF12 were partially purified. On the basis of these biochemical and genetic results, it is suggested that structural alterations in the primary sequence of the y subunit corresponding to residues 1 to 167 cause more extensive defects in the assembly of F1 than alteration in the sequence of residues 168 to 287. The proton-translocating ATPase of E.scherichia coli catalyzes the hydrolysis and synthesis of ATP reversibly. The enzyme is composed of two distinct portions, F1 and FO," which are extrinsic and intrinsic portions, respectively, of the membranes. F1 has catalytic activity and has five different subunits, CX, p, y, 6, and t (l
Journal of Biological Chemistry
The proton-translocating ATPase complex (F1F0) of Escherichia coli was purified after inductin of a lambda-transducing phage (lambda asn5) carrying the ATPase genes of th unc operon. ATPase activity of membranes prepared from the induced lambda-unc lysogen was 6-fold greater than the activity of membranes prepared from strains lacking the unc-transducing phage, confirming the report of Kanazawa et al. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 1126-1130). The F1F0-ATPase complex was purified in comparable yield from either enriched membranes or control membranes using a modification of the procedure reported by Foster and Fillingame ((1979) J. Biol. Chem. 254, 8230-8236). EAch of the eight subunits that had been reported as components of the F1F0 complex from wild type E. coli was overproduced in the lambda-unc lysogen. All eight subunits co-purified in the same stoichiometric proportion as in the complex purified from wild type E. coli. We conclude that all eight subunits are likel...
Advances in biophysics, 1987
Recent results on ATPase, mainly from E. coli, obtained by biochemical and molecular biological approaches are reviewed, with special emphasis on results obtained in this laboratory. The advantages of using E. coli in studies of this important enzyme in oxidative phosphorylation are indicated: variant enzymes with specific amino acid replacements can be obtained and their functions and structures can be compared with those of the wild-type enzyme. Structural aspects of this complex enzyme are discussed, including the primary amino acid sequences and molecular assembly of subunits, and mechanistic aspects of the catalytic mechanism and proton translocation.
Archives of Biochemistry and Biophysics, 1987
Mutations in the uncA gene of Escherichia coli cause loss of both oxidative phosphorylation and ATP-driven generation of the transmembrane proton gradient. The uncA gene encodes the a-subunit of the Fi-sector of the E. coli membrane proton-ATPase. F1-a-subunit from normal (uric+) E. coli binds ATP tightly (Ko = 0.1 PM) and undergoes a large ATP-induced conformational change, but the functional role of the ATP-binding site is currently unknown. There is disagreement in the literature as to whether the ATP-binding site is present or lacking in F1-a-subunit from uncA mutant strains. One obstacle in studying this question is the difficulty of purifying mutant a-subunits in native form. In order to circumvent this difficulty we have studied ATP binding and ATP-induced conformational changes in mixtures of F1 subunits obtained by dissociating uncA mutant Fi. Anti-a antibody was used in conjunction with immunoblotting to identify the a-subunits in the mixtures. Retention of native conformation by the (Ysubunits was demonstrated by the fact that the dissociated a-subunits were fully competent to repolymerize with other F1 subunits to yield intact F1 aggregate. The results show that, contrary to previous reports, a-subunits from three catalytically defective uncA mutants do indeed bind ATP and do undergo an ATP-induced conformational change. The binding affinity of a-subunit for ATP was lower than normal in each of the three mutants, but this is not likely to be a significant factor under physiological conditions. 0 1987 Academic Press, Inc. ing of ATP to the a-subunit conferred enhanced resistance to trypsin proteolysis, and that the required concentrations of 309