Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Investigations have been made of the kinetic effects of the antibiotic aurovertin on the ATPase and ITPase activity of isolated rat liver mitochondrial ATPase. Unusual patterns of inhibition, decreasing slope, and increasing y-intercept values of double reciprocal plots, were observed with Mg-ATP as the substrate under various conditions. Under specified conditions, aurovertin stimulated hydrolysis of MgATP. The inhibition of MgITP hydrolysis was uncompetitive. Aurovertin eliminated the HCO3-minus stimulation of MgATP hydrolysis. The implications of these findings for the mechanism of mitochondrial ATPase are briefly discussed.
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PMID:Influence of aurovertin on mitochondrial ATPase activity. 12 77

The bovine heart mitochondrial F1-ATPase (MF1) is inactivated by 5'-p'-fluorosulfonylbenzoyl-8-azidoadenosine (8-N3-FSBA) with an apparent Kd of 0.47 mM at pH 8.0 and 23 degrees C in the absence of light. Irradiation of dark-inactivated enzyme with long-wavelength UV light produced cross-linked dimers and, to a lesser extent, trimers made up of alpha and beta subunits. Two major radioactive peptides were resolved by high-performance liquid chromatography from tryptic digests of MF1 which had been inactivated with 8-N3-FSB[3H]A at pH 8.0 in the dark. Sequence analysis revealed that one contained Tyr-beta 368 and the other contained His-beta 427 which were labeled in the ratio of 18:15. Sequence analysis of radioactive tryptic peptides isolated from digests of irradiated MF1 derivatized with 8-N3-FSB[3H]A showed that photolysis induced cross-linking of His-427 to Tyr-345 within the same beta subunit in high yield. When MF1 derivatized with 8-N3-FSB[3H]A was irradiated in the presence of beta-mercaptoethanol, alpha-beta cross-links were eliminated, whereas those between His-beta 427 and Tyr-beta 345 were unaffected. Analysis of radioactive peptides in tryptic digests of MF1 derivatized with 8-N3-FSB[3H]A and then irradiated in the presence or absence of beta-mercaptoethanol showed that the nitrene generated from reagent attached to Tyr-beta 368 participates in formation of alpha-beta cross-links in the absence of beta-mercaptoethanol. Therefore, the nitrene generated from reagent tethered to His-beta 427 is shielded from solvent and reacts with the side chain of Tyr-beta 345. In contrast, the nitrene generated from reagent attached to Tyr-beta 368 is exposed to solvent, but in the absence of scavengers reacts with side chains present in the alpha subunit. Irradiation of MF1, partially inactivated with 8-N3-FSBA, led to loss of residual ATPase activity without affecting residual ITPase activity. The amount of photoinactivation was greater when partial dark inactivation was performed at pH 6.9, where modification of His-beta 427 predominates, than when performed at pH 8.0, where modification of Tyr-beta 368 predominates. This suggests that cross-linking of His-beta 427 to Tyr-beta 345, and not cross-linking of alpha and beta subunits, is responsible for the augmented inactivation induced by irradiation.
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PMID:Irradiation of the bovine mitochondrial F1-ATPase previously inactivated with 5'-p-fluorosulfonylbenzoyl-8-azido-[3H]adenosine cross-links His-beta 427 to Tyr-beta 345 within the same beta subunit. 132 8

The bovine heart mitochondrial F1-ATPase (MF1) is inactivated by 5'-p-fluorosulfonylbenzoylethenoadenosine (FSB epsilon A) with pseudo-first order kinetics. The dependence of the rate of inactivation on the concentration of FSB epsilon A revealed an apparent Kd of 0.25 mM. ATP and ADP, and to a lesser extent, ITP and IDP provide partial protection against inactivation by the reagent. Isolation and sequence analysis of major radioactive fragments in peptic or cyanogen bromide digests of MF1 inactivated with [3H]FSB epsilon A indicate that modification of Tyr-alpha 244 is associated with the loss of activity observed. Assessment of the amount of Tyr-alpha 244 derivatized with [3H]FSB epsilon A at specific points during inactivation of the ATPase indicates that maximal inactivation is achieved on modification of this residue in slightly greater than one copy of the alpha subunit. The following characteristics of inactivation of MF1 by FSB epsilon A have also been determined. (a) The rate of inactivation of ITPase activity by FSB epsilon A is 1.4 times greater than that observed for inactivation of ATPase activity under identical conditions. (b) After maximally inactivating the capacity of MF1 to hydrolyze saturating ATP with FSB epsilon A, the modified enzyme retained its capacity to hydrolyze substoichiometric ATP. (c) Inactivation of the ATPase by FSB epsilon A is accelerated by Pi. In each of the above characteristics, MF1 modified by FSB epsilon A resembles enzyme inactivated with 5'-p-fluorosulfonylbenzoyladenosine (FSBA) more than it does enzyme inactivated with 5'-p-fluorosulfonylbenzoylinosine (FSBI). Furthermore, prior inactivation of MF1 with FSBA completely prevents labeling of Tyr-alpha 244 with [3H]FSB epsilon A, whereas prior inactivation of the enzyme with FSBI does not. Since a single catalytic site is modified when FSBI inactivates MF1 whereas three noncatalytic sites are modified when it is maximally inactivated with FSBA, it is concluded that FSB epsilon A also modifies noncatalytic sites.
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PMID:Tyrosine alpha 244 is derivatized when the bovine heart mitochondrial F1-ATPase is inactivated with 5'-p-fluorosulfonylbenzoylethenoadenosine. 213 76

Phenotypic revertants have been selected from mutants of the yeast Schizosaccharomyces pombe devoid of either alpha or beta subunits of mitochondrial ATPase-ATPsynthase. In contrast to parental mutants, phenotypic revertants are able to grow on glycerol respiratory medium and show immunodetectable alpha and beta subunits. However, growth and cellular respiration are only partially restored as compared to the wild strain, indicating that the recovered subunits are mutated. ATPase activity of revertant submitochondrial particles shows markedly different parameters: more acidic optimal pH, absence of bicarbonate activation and decreased sensitivity to azide inhibition in the alpha subunit-modified R3.51. Opposite differences are observed in the beta subunit-modified R4.3: more alkaline optimal pH, much higher bicarbonate activation, and increased sensitivity to azide. The ITPase activity of R4.3 submitochondrial particles is also more sensitive to azide as compared to the wild strain. ATPase activity of purified F1 also exhibits marked differences: loss of bicarbonate-sensitive negative cooperativity, decreased sensitivity to both ADP and azide inhibitions in the R3.51 revertant. On the contrary, increased negative cooperativity and increased sensitivity to both ADP and azide inhibitions are observed for the R4.3 revertant enzyme which in addition exhibits a much lower maximal rate. The beta subunit-mutation of R4.3 also increases the sensitivity of ITPase activity to tripolyphosphate inhibition, whereas the alpha subunit-mutation of R3.51 is without any effect. Soluble F1 with beta subunit-mutation is very sensitive to high ammonium sulfate concentrations required for enzyme precipitation and concentration and known to partially deplete the enzyme from its endogenous nucleotides. On the contrary, poly(ethylene)glycol is very efficient for preparing from any strain a pure and very stable enzyme retain-ing high amounts of endogenous nucleotides. The R4.3 revertant F1 retains even more nucleotides than the wild-strain F1 and is much less sensitive to high iodide concentrations which favor enzyme dissociation and precipitation. The tryptophan intrinsic fluorescence of F1 is modified by both mutations that increase the maximal emission intensity. The most important effect is produced by beta subunit-mutation which decreases the quenchable fraction, one-third to one-half tryptophans being no longer accessible to iodide. The overall results suggest that both mutations modify enzyme-nucleotide interactions: the alpha subunit-mutation of R3.51 would favor ADP release by lowering interactions with the adenine moiety, whereas the beta subunit-mutation of R4.3 would lower ADP release by strengthening interactions with the phosphate chain moiety.
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PMID:Structure-function relationships of mitochondrial ATPase-ATPsynthase using Schizosaccharomyces pombe yeast mutants with altered F1 subunits. 252 9

This study examined the nature of the metal-nucleotide complexes which serve as substrates, products, and intermediates in the beef heart mitochondrial ATPase reaction. The two methods employed involved the use of phosphorothioate ATP analogs as substrates in the presence of Mg2+ or Cd2+ and the use of substitution inert Cr X ATP complexes (the isolated diastereomers of the bidentate complexes) along with the newly synthesized Cr X ITP complexes as inhibitors of both the F1-ATPase and F1-ITPase activities. Little stereoselectivity was observed in the inhibition of F1-ATPase and F1-ITPase activities by the isolated diastereomers of beta,gamma-bidentate CrATP, while the inhibition by the delta,alpha,beta-bidentate CrADP diastereomer was greater than that of the lambda epimer. gamma-Monodentate CrITP was a weak inhibitor of both the ATPase and ITPase activities, whereas beta,gamma-bidentate CrITP failed to show any inhibition at all up to a concentration of 3.2 mM. When adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) was used as the substrate, (VmSp]/(Vm(Rp] with Mg2+ present was 2.7 at 31 degrees C and 3.5 at 13 degrees C. The (Vm/Km(Sp]/(Vm/Km(Rp] ratios with Mg2+ present were 15.3 at 31 degrees C and 73.3 at 13 degrees C. With Cd2+ present, the (Vm(Sp]/(Vm(Rp] ratios were 0.81 and 0.65 at 31 and 13 degrees C, respectively. The (Vm/Km(Sp]/(Vm/Km(Rp] ratios with Cd2+ present were 1.17 at 31 degrees C and 1.34 at 13 degrees C. The large activation energy observed for the isomers of CdATP beta S was not observed for MgATP beta S, MgATP, or CdATP. The Vm for Cd adenosine 5'-O-thiotriphosphate (ATP gamma S) hydrolysis was the largest of all the metal-phosphorothioate nucleotide complexes, while that for MgATP gamma S was the smallest. The results are interpreted in terms of a catalytic model for F1-catalyzed nucleotide hydrolysis describing metal-nucleotide chelation during the reaction.
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PMID:Metal-nucleotide structural characteristics during catalysis by beef heart mitochondrial F1. 286 Jan 7

A soluble Mg-dependent ATPase, similar to the mitochondrial ATPase from beef heart, has been isolated from heart mitochondria of salmon (Salmo salar). The salmon heart ATPase has 5 subunits with molecular weights similar to the beef heart enzyme, but the Stoke's radius of the intact salmon enzyme is larger. The salmon heart ATPase is less temperature labile than the beef heart enzyme. The salmon heart ATPase is strongly inhibited by ADP, and the inhibition is highly temperature dependent. The ITPase activity is also inhibited by IDP (Ki = 180 micron). 2,4-Dinitrophenol in small concentrations stimulates the ITPase activity as well as the ATPase activity of the "washed" salmon heart enzyme. However, in an enzyme preparation which had been freed of most of the bound nucleotides by dialysis in the presence of glycerol (Roveri et al., 1980) the ITPase activity is not stimulated by 2,4-dinitrophenol.
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PMID:Some properties of isolated mitochondrial ATPase from salmon heart. 615 Aug 4

The mono- and bidentate forms of adenosine 5'-diphosphate, chromium (III) salt (CrADP) were separated using Sephadex G-10 column chromatography. The isomeric purity of the two forms was monitored using high voltage electrophoresis and column chromatography. The same techniques were employed to assess the purity of the mono-, bi-, and tridentate forms of adenosine 5'-triphosphate, chromium (III) salt (CrATP). Distinct differences in the interaction of beef heart mitochondrial ATPase with the various isomers of chromium nucleotides were seen in kinetic studies. Monodentate CrADP was a competitive inhibitor of the ATP hydrolysis activity of both purified ATPase and submitochondrial particles. However, when ITPase activity was examined, noncompetitive inhibition was observed. The bidentate isomer of CrADP did not affect ATPase activity. Enzymatic synthesis of the transition state analog of ATP synthesis and hydrolysis, Pi-CrADP occurred exclusively with the monodentate isomer of CrADP. It was also found that only the mono- and tridentate forms of CrATP were potent inhibitors of ATP hydrolysis by beef heart mitochondrial ATPase. These results are discussed in terms of possible ATP synthesis and hydrolysis mechanisms.
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PMID:Structural preferences for the binding of chromium nucleotides by beef heart mitochondrial ATPase. 645 68

Lineweaver-Burk plots for ATP hydrolysis catalyzed by bovine heart mitochondrial F1-ATPase (MF1) at 30 degrees C are biphasic, whereas they are linear at 15 degrees C. The rate of inactivation of the enzyme at 23 degrees C by 5'-[(p-fluorosulfonyl)benzoyl]adenosine (FSBA), which derivatizes noncatalytic nucleotide binding sites, is about 4 times faster when loss of activity is monitored at 15 degrees C as opposed to 30 degrees C. This suggests that maximal loss of ATPase monitored at 15 degrees C is observed when a single noncatalytic site is derivatized, whereas maximal inactivation at 30 degrees C requires modification of three noncatalytic sites. Prior incubation of MF1 depleted of endogenous nucleotides (nd-MF1) with pyrophosphate (PPi) stimulates ATPase activity 2-fold when assayed at 30 degrees C and pH 8.0. This stimulation correlates with binding of [32P]PPi to the second and third binding sites for PPi to be filled. Prior binding of PPi to nd-MF1 increases the rate of inactivation of the enzyme by FSBA at 23 degrees C about 4-fold when loss of activity is monitored at 30 degrees C and pH 8.0, whereas it does not affect the rate of inactivation when loss of ATPase is monitored at 15 degrees C or loss of ITPase is monitored at 30 degrees C. This indicates that the accelerated rate of inactivation induced by PPi when assays are conducted at 30 degrees C is not due to an increased rate of derivatization of noncatalytic sites. After 85% inactivation with FSBA, nd-MF1 retains the capacity to bind 2.8 mol of [32P]PPi per mole.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Lowered temperature or binding of pyrophosphate to sites for noncatalytic nucleotides modulates the ATPase activity of the beef heart mitochondrial F1-ATPase by decreasing the affinity of a catalytic site for inhibitory MgADP. 799 54

The mechanism of action of F(1)F(0)-ATP synthase is controversial. Some favor a tri-site mechanism, where substrate must fill all three catalytic sites for activity, others a bi-site mechanism, where one of the three sites is always unoccupied. New approaches were applied to examine this question. First, ITP was used as hydrolysis substrate; lower binding affinities of ITP versus ATP enable more accurate assessment of sites occupancy. Second, distributions of all eight possible enzyme species (with zero, one, two or three sites filled) as fraction of total enzyme population at each ITP concentration were calculated, and compared with measured ITPase activity. Confirming data were obtained with ATP as substrate. Third, we performed a theoretical analysis of possible bi-site mechanisms. The results argue convincingly that bi-site hydrolysis activity is negligible, and may not even exist. Effectively, tri-site hydrolysis is the only mechanism. We argue that only tri-site hydrolysis drives subunit rotation. Theoretical analyses of possible bi-site mechanisms reveal serious flaws, not previously recognized. One is that, in bi-site catalysis, the predicted direction of subunit rotation is the same for both ATP synthesis and hydrolysis; a second is that infrequently occurring enzyme species are required.
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PMID:Bi-site catalysis in F1-ATPase: does it exist? 1145 60

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