Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.3.14 (ATP synthase)
 7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The alpha and beta subunits of F1-ATPase are homologous in primary structure and have similar folding topologies. The position of the essential Glu residue in the catalytic sites which reside in the beta subunits is occupied by a Gln residue in the noncatalytic nucleotide binding sites which reside in the alpha subunits. To test if an exchange of catalytic and noncatalytic binding sites is possible, we have replaced the Gln-Lys sequence in the noncatalytic binding site of the alpha subunit with Glu-Arg and, reciprocally, the Glu in the catalytic site of the beta subunit with Gln. The resultant mutant alpha3beta3gamma complex lost steady-state ATPase activity. However, HPLC analysis of tryptic digests of the mutant alpha3beta3gamma complex which had been photolabeled with 2-N3-[8-3H]ATP revealed that ATP tethered to the noncatalytic binding side was hydrolyzed, indicating that a primitive catalytic ability was generated at the alpha subunit by the introduced Glu.
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PMID:An attempt to convert noncatalytic nucleotide binding site of F1-ATPase to the catalytic site: hydrolysis of tethered ATP by mutated alpha subunits in the enzyme. 860 64

Incubation of chloroplast thylakoids with pyridoxal 5'-phosphate for a short time (5 s) modified the lysine residues of the gamma subunit of ATP synthase. Energization of thylakoids by illumination increased the reactivity of Lys24 by a factor of three and decreased the reactivity of Lys30 to 60%. The reactivities of these residues reached their maximum and minimum values, respectively, within 1 s after the onset of illumination. Illumination of thylakoids increased the reactivities of Lys222 and Lys231 in two steps by a factor of three. The first step was completed within 1 s and the second step was completed 20-30 s after the onset of illumination. In the presence of 10 mM NH4Cl, illumination of thylakoids did not change the reactivities of these lysine residues. These results suggest that the Lys24- and Lys30-containing region of the gamma subunit changes its conformation rapidly in response to delta mu H+ and that the Lys222- and Lys231-containing region of the gamma subunit changes its conformation in two steps in response to delta mu H+ formation.
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PMID:Energizing effects of illumination on the reactivities of lysine residues of the gamma subunit of chloroplast ATP synthase. 861 18

Subunit interactions among the F1-ATPase subunits were studied by the yeast two-hybrid system. Various pairwise combinations of genes encoding alpha, beta, gamma, delta and epsilon subunits of Escherichia coli H+-ATPase fused to the DNA-binding or activation domain of the yeast GAL4 gene were introduced into yeast and expression of a reporter gene encoding beta-galactosidase was detected. Combinations of the alpha and beta subunit genes, and of the epsilon and gamma subunit genes showed high levels of reporter gene expression, while those of alpha and delta, beta and delta, gamma and delta, and delta and epsilon demonstrated weak but significant reporter gene expression. However, combinations of alpha and gamma, beta and gamma, alpha and epsilon, and beta and epsilon did not induce reporter expression. None of the fused genes alone induced reporter gene expression. These results suggested that specific and strong interactions between the alpha and beta, gamma and epsilon, and weak interactions between the alpha and delta, beta and delta, and gamma and delta subunits occurred in yeast cells in the two-hybrid system. Effects of previously identified mutant beta subunits with Leu-40 to Pro. Glu-41 to Lys or Pro-332 to Gln substitutions which caused defects in molecular assembly of F1-ATPase were analyzed with regard to alpha-beta interactions. No interaction of the alpha and beta subunits was observed in this system using the beta subunit with mutation of Pro-332 to Gln. However, for the other two mutations, alpha-beta interactions were observed. This system may be useful for isolating mutants which have defects in interaction of F1-ATPase subunits.
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PMID:Interactions of the F1-ATPase subunits from Escherichia coli detected by the yeast two-hybrid system. 864 96

Three intragenic second-site suppressors, P353L, T237I, and L390F, were identified that suppressed two mutations in, and one adjacent to, the P-loop in the beta-subunit of the yeast F1-ATPase. The crystal structure of bovine F1-ATPase (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628) shows that these suppressor residues are located in the nucleotide-binding domain. Specific hypotheses have been formulated that suggest the conformational coupling of the P-loop with the suppressor sites. P353L is in a "catch" region, which forms unique interactions with the gamma-subunit in the three different conformational states of the catalytic site. The identification of this suppressor mutation demonstrates genetically that the catch region is conformationally coupled to the P-loop. T237I is shown to interact with Lys-209, which occurs just after the P-loop. This suggests that this interaction changes the conformation of the P-loop to suppress the initial mutation. L390F interacts with Ala-181, which is adjacent to the P-loop. The mechanism of this suppression is suggested to occur through the interactions of L390F with Ala-181. These results identify critical interactions that modulate the structure of the P-loop and thus the biochemistry of the enzyme.
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PMID:Intragenic suppressors of P-loop mutations in the beta-subunit of the mitochondrial ATPase in the yeast Saccharomyces cerevisiae. 866 32

The delta-subunit of bovine F1-ATPase was expressed from a bacterial vector at fairly high level in Escherichia coli, but the yield of bovine epsilon-subunit was rather low under similar conditions. However, co-expression of the proteins from a dicistronic operon delta-epsilon in the same expression vector, produced both of them in good yield in a soluble form in the bacterial cytoplasm, and by chromatography it was found that the delta- and epsilon-subunits were associated in a stable complex. The amino groups in the complex were labelled exhaustively by chemical reaction under denaturing conditions with ethyl-[1-14C]acetimidate. The alpha-amino groups of the proteins were unmodified, but complete reaction of all epsilon-amino groups in both proteins was demonstrated by determination of the molecular masses of the modified proteins by electrospray MS. The modified subunits were separated by denaturing gel electrophoresis, and from measurements of the ratio of incorporated radioactivities and the lysine contents of the proteins, it was calculated that the subcomplex contains equimolar amounts of the two proteins. As the apparent molecular mass of the complex determined by gel filtration was 29 kDa, it appears that the complex contains one copy of each protein. It is likely that the delta- and epsilon subunits are associated in a similar manner in the bovine F1-ATPase complex, and that, like a bacterial homologue of the delta-subunit, they interact with the gamma- and beta-subunits.
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PMID:The delta- and epsilon-subunits of bovine F1-ATPase interact to form a heterodimeric subcomplex. 867 87

Subunit 8 (Y8) is a component of the proton channel of yeast (Saccharomyces cerevisiae) mitochondrial ATP synthase (mtATPase), whose function in the complex remains to be precisely defined. Y8 variants truncated at residue 46 (Lys47-->STP), or in which each of three conserved C-terminal amino acid residues (Arg37, Arg42 and Lys47) were substituted with isoleucine, have defects in assembly and function. The additional positive charge substitution (Gln29-->Lys) was introduced into each of the variants to determine whether functional compensation for these defects could be achieved. In the case of the (Lys47-->STP) variant, the additional positive charge restored the ability of cells to grow on non-fermentable substrate. By contrast, for the (Lys47-->Ile) variant the additional positive charge did not confer any improvement in cellular growth rate compared to that of cells expressing the Lys47-->Ile substitution alone. For the (Arg42-->Ile) and (Arg37-->Ile) variants, the presence of the Gln29-->Lys substitution failed to restore growth of host cells lacking endogenous subunit 8 on non-fermentable substrate. However, use of an in vitro assembly assay revealed that, unlike their respective parents (Arg42-->Ile or Arg37-->Ile), the (Gln29-->Lys Arg42-->Ile) and (Gln29-->Lys Arg37-->Ile) variants assemble into mtATPase. Thus we conclude that Arg42 and Arg37 have a role in mtATPase function, in addition to being required for assembly of Y8 into mtATPase.
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PMID:Non-functional variants of yeast mitochondrial ATP synthase subunit 8 that assemble into the complex. 879 51

Peptide segments of the inhibitor protein (IF1) of the F0F1 ATP synthase complex from bovine-heart mitochondria have been constructed by chemical synthesis. The IF1-(42-58)-peptide was equally effective as IF1 in inhibiting the ATPase activity of both the F0F1 complex in the mitochondrial membrane deprived of IF1 (SMP) and soluble F1. The IF1-(22-46)-peptide inhibited the ATPase activity in the soluble F1 but had no effect on either the ATPase activity or H+ conduction in SMP. Substitution of the His or Lys residues with Ala in the IF1-(42-58)-peptide decreased the inhibition of ATP hydrolysis. The inhibition exerted by the IF1-(42-58)-peptide on ATP hydrolysis in SMP exhibited a pH dependence, similar to that observed with IF1, which was lost upon replacement of His or Lys with Ala. In soluble F1, inhibition of ATP hydrolysis by IF1, the IF1-(42-58)-peptide and the IF1-(22-46)-peptide was pH dependent when F1 was first incubated with ATP. The IF1-(42-58)-peptide also caused inhibition of passive H+ conduction in SMP. This activity of the synthetic peptide was weaker, as compared to that of IF1, and practically unaffected by substitution of His or Lys with Ala. An antibody against the IF1-(42-58)-synthetic peptide stimulated ATP hydrolysis in the membrane-bound F0F1 complex with associated IF1 but was without effect on H+ conduction. An antibody against IF1 stimulated both processes.
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PMID:Identification of functional domains and critical residues in the adenosinetriphosphatase inhibitor protein of mitochondrial F0F1 ATP synthase. 884 13

Batten disease, or juvenile neuronal ceroid-lipofuscinosis, is an autosomal-recessive hereditary disorder that leads to blindness, severe neurological degeneration, and premature death. The disease is characterized by massive accumulation of lysosomal storage bodies in most tissues. A significant constituent of the storage material is a protein that appears to be almost identical to a small hydrophobic inner mitochondrial membrane protein, subunit c of ATP synthase. The protein isolated from the storage bodies contains an epsilon-N-trimethyl-L-lysine (TML) residue at amino acid position 43. The presence of TML in the stored protein suggests that one of the lysine residues in subunit c is normally trimethylated, and this trimethylation may act as a signal to initiate degradation of the protein. Free TML produced by the degradation of TML-containing proteins is the first intermediate in the carnitine biosynthetic pathway. It is possible that trimethylated subunit c is a major source of the free TML used in carnitine biosynthesis. If this is the case, one would predict that the genetic defect resulting in the accumulation of TML containing subunit c would also reduce systemic levels of free TML and carnitine. To evaluate this possibility, plasma TML and carnitine levels were measured in affected human subjects, heterozygous carriers, and normal controls. Both TML and carnitine levels were significantly depressed in the affected individuals. This suggests that subunit c is normally a major source of TML for carnitine biosynthesis. In Batten disease, failure to degrade the TML-containing form of subunit c is probably responsible for the reduction in plasma TML and carnitine levels.
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PMID:Decreased plasma carnitine and trimethyl-L-lysine levels associated with lysosomal accumulation of a trimethyl-L-lysine containing protein in Batten disease. 898 35

Replacement of the F0F1 ATP synthase gamma subunit Met-23 with Lys (gammaM23K) perturbs coupling efficiency between transport and catalysis (Shin, K., Nakamoto, R. K., Maeda, M., and Futai, M. (1992) J. Biol. Chem. 267, 20835-20839). We demonstrate here that the gammaM23K mutation causes altered interactions between subunits. Binding of delta or epsilon subunits stabilizes the alpha3beta3gamma complex, which becomes destabilized by the mutation. Significantly, the inhibition of F1 ATP hydrolysis by the epsilon subunit is no longer relieved when the gammaM23K mutant F1 is bound to F0. Steady state Arrhenius analysis reveals that the gammaM23K enzyme has increased activation energies for the catalytic transition state. These results suggest that the mutation causes the formation of additional bonds within the enzyme that must be broken in order to achieve the transition state. Based on the x-ray crystallographic structure of Abrahams et al. (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the additional bond is likely due to gammaM23K forming an ionized hydrogen bond with one of the betaGlu-381 residues. Two second site mutations, gammaQ269R and gammaR242C, suppress the effects of gammaM23K and decrease activation energies for the gammaM23K enzyme. We conclude that gammaM23K is an added function mutation that increases the energy of interaction between gamma and beta subunits. The additional interaction perturbs transmission of conformational information such that epsilon inhibition of ATPase activity is not relieved and coupling efficiency is lowered.
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PMID:Energy coupling, turnover, and stability of the F0F1 ATP synthase are dependent on the energy of interaction between gamma and beta subunits. 899 37

Three critical residues, beta-Lys-155, beta-Asp-242, and beta-Glu-181, situated close to the gamma-phosphate of MgATP in F1-ATPase catalytic sites, were investigated. The mutations betaK155Q, betaD242N, and betaE181Q were each combined with the betaY331W mutation; the fluorescence signal of beta-Trp-331 was used to determine MgATP, MgADP, ATP, and ADP binding parameters for the three catalytic sites of the enzyme. The quantitative contribution of side chains to binding energy at all three catalytic sites was calculated. The following conclusions were made. The major functional interaction of beta-Lys-155 is with the gamma-phosphate of MgATP and is of primary importance at site 1 (the site of highest affinity) and site 2. Release of MgATP during oxidative phosphorylation requires conformational re-positioning of this residue. The major functional interaction of beta-Asp-242 is with the magnesium of the magnesium nucleotide at site 1; it has little or no influence at site 2 or 3. In steady-state turnover, the MgATP hydrolysis reaction occurs at site 1. beta-Glu-181 contributes little to nucleotide binding; its major catalytic effect derives apparently from a role in reaction chemistry per se. This work also emphasizes that nucleotide binding cooperativity shown by the three catalytic sites toward MgATP and MgADP is absolutely dependent on the presence of magnesium.
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PMID:F1-ATPase, roles of three catalytic site residues. 901 18


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