Evidence for functional heterogeneity among the catalytic sites of the bovine heart mitochondrial F1-ATPase.

Bullough, David A.; Verburg, John G.; Yoshida, Masasuke; Allison, William S.

doi:10.1016/s0021-9258(18)60863-4

Cited by 58 publications

(8 citation statements)

References 42 publications

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“…Consistent with this finding, Chernyak and Cross (36) reported that inhibitory Mg(2-N 3 -ADP) was entrapped in a single catalytic site of MF 1 after adding stoichiometric 2-N 3 -ATP to it in the presence of Mg 2+ in the dark. Furthermore, it has been demonstrated that considerable [R-32 P]ADP fails to release when substoichiometic [R, γ-32 P]-ATP is hydrolyzed at a single catalytic site of MF 1 (37). In contrast, Milgrom and Cross (38) have recently reported that product ADP and Pi dissociate at nearly equal rates (about 5.5 × 10 -3 s -1 ) after adding substoichiometric ATP to nucleotide-depleted MF 1 followed by diluting the enzyme 667-fold in buffer plus 1.1 mg mL -1 BSA.…”

Section: T H Imentioning

confidence: 99%

“…Release of products was assessed by passing samples of diluted enzyme through centrifuge columns of Sephadex equilibrated with the same buffer containing 1.1 mg mL -1 BSA. In the studies where slow dissociation of ADP was observed, slow release of ADP was assessed by enzyme assay (35,36), by enzyme assay and quenching of tryptophan fluorescence of the R 3 -(βY 341 W) 3 γ subcomplex of TF 1 , and by passing samples through centrifuge columns in the absence of BSA (37). Consideration of these differences suggests that BSA might influence the rate of dissociation of ADP when MgATP is hydrolyzed at a single catalytic site.…”

Section: T H Imentioning

confidence: 99%

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The α₃(βY₃₄₁W)₃γ Subcomplex of the F₁-ATPase from the Thermophilic Bacillus PS3 Fails to Dissociate ADP When MgATP Is Hydrolyzed at a Single Catalytic Site and Attains Maximal Velocity When Three Catalytic Sites Are Saturated with MgATP^†

1998

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The hydrolytic properties of the alpha3beta3gamma and mutant alpha3(betaY341W)3gamma subcomplexes of the TF1-ATPase have been compared. ATPase activity of the mutant is less sensitive to turnover-dependent inhibition by azide, less suppressed by increasing concentrations of Mg2+ during assay, and less stimulated by lauryl dimethylamine oxide (LDAO). Therefore, it has much lower propensity than wild-type to entrap inhibitory MgADP in a catalytic site during turnover. The fluorescence of the introduced tryptophans in the alpha3(betaY341W)3gamma subcomplex is completely quenched when catalytic sites are saturated with ATP or ADP with or without Mg2+ present. As reported for the betaY331W mutant of Escherichia coli F1 (Weber, J., Wilke-Mounts, S., Lee, R. S.-F., Grell, E., Senior, A. E. (1993) J. Biol. Chem. 268, 20126-20133), this provides a direct probe of nucleotide binding to catalytic sites. Addition of stoichiometric MgATP to the mutant subcomplex quenched one-third the tryptophan fluorescence which did not recover after 60 min. This was caused by entrapment of MgADP in a single catalytic site. Titration of catalytic sites of the alpha3(betaY341W)3gamma subcomplex with MgADP or MgATP revealed Kd's of < 50 nM, about 0.25 microM and about 35 microM. Titrations were not affected by azide, whereas LDAO lowered the affinities of catalytic sites 2 and 3 for MgADP by 5-fold and 2-fold, respectively. During titration with MgATP, LDAO slightly lowered affinity at ATP concentrations below 30 microM and had no effect at ATP concentrations above 30 microM. Maximal velocity was attained when the third catalytic site was titrated with MgATP in the presence or absence of LDAO. The same Kd's for binding MgATP to the (alphaA396C)3beta3(gammaA22C) mutant were observed before and after inactivating it by cross-linking alpha to gamma. This implies that the different affinities of catalytic sites for MgATP do not represent negative cooperativity, but rather represent heterogeneous affinities of catalytic sites dictated by the position of the coiled-coil of the gamma subunit within the central cavity of the (alpha beta)3 hexamer.

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Section: T H Imentioning

confidence: 99%

Section: T H Imentioning

confidence: 99%

The α₃(βY₃₄₁W)₃γ Subcomplex of the F₁-ATPase from the Thermophilic Bacillus PS3 Fails to Dissociate ADP When MgATP Is Hydrolyzed at a Single Catalytic Site and Attains Maximal Velocity When Three Catalytic Sites Are Saturated with MgATP^†

1998

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“…unisite catalysis by bovine heart ATP synthase in membranes has similar characteristics to that of soluble Fh and in response to questions regarding the significance of unisite catalysis in mitochondrial F, (Bullough et al, 1987), Penefsky (1988) and Cunningham and Cross (1988) demonstrated that unisite catalysis and its "promotion" to a multisite rate are a reflection of activity at a normal catalytic site. Unisite catalysis has been confirmed in Fj-ATPases from other sources including Escherichia coli (Wise et al, 1984) and yeast (Mueller, 1990), and Penefsky and Cross (1991) The significance of the discovery of unisite catalysis and of determination of its associated rate and equilibrium constants is severalfold, (a) It complements, supports, and extends the "binding change" model for ATP synthesis (Boyer, 1989).…”

mentioning

confidence: 97%

Catalytic sites of Escherichia coli F1-ATPase. Characterization of unisite catalysis at varied pH

Al‐Shawi¹,

Senior²

1992

Biochemistry

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Using manual rapid-mixing procedures in which small, equal volumes of Escherichia coli F1-ATPase and [gamma-32P]ATP were combined at final concentrations of 2 and 0.2 microM, respectively (i.e., unisite catalysis conditions), it was shown that greater than or equal to 66% of the 32P became bound to the enzyme, with the ratio of bound ATP/bound Pi equal to 0.4 and the rate of dissociation of bound [32P]Pi equal to 3.5 x 10(-3) s-1, similar to previously published values. Azide is known to inhibit cooperative but not unisite catalysis in F1-ATPase [Noumi, T., Maeda, M., & Futai, M. (1987) FEBS Lett. 213, 381-384]. In the presence of 1 mM sodium azide, 99% of the 32P became bound to the enzyme, with the ratio of bound ATP/bound Pi being 0.57. These experiments demonstrated that when conditions are used which minimize cooperative catalysis, most or all of the F1 molecules bind substoichiometric ATP tightly, hydrolyze it with retention of bound ATP and Pi, and release the products slowly. The data justify the validity of previously published rate constants for unisite catalysis. Unisite catalysis in E. coli F1-ATPase was studied at varied pH from 5.5 to 9.5 using buffers devoid of phosphate. Rate constants for ATP binding/release, ATP hydrolysis/resynthesis, Pi release, and ADP binding/release were measured; the Pi binding rate constant was inferred from the delta G for ATP hydrolysis. ATP binding was pH-independent; ATP release accelerated at higher pH. The highest KaATP (4.4 x 10(9) M-1) was seen at physiological pH 7.5.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…When the ratio between substrates and ATP synthase is less than one, catalysis has been shown to occur at the high-affinity site, both in synthesis and hydrolysis direction-the so-called "unisite" catalysis, which is several orders of magnitude slower than multisite steady-state catalysis (Penefsky and Cross, 1991). Though unisite catalysis has often been considered a step cooperatively integrated to each turnover of the multisite catalysis, it has also been shown to take place in the absence of rotation (García and Capaldi, 1998), a result in agreement with those of Bullough et al (1987), and with a recent cryo-EM study, which indicates that unisite catalysis is an initial reaction that is distinguished from steady-state rotary catalysis (Nakano et al, 2022). In addition, Sakaki et al (2005) measured the same rotation rates from mM down to nM ATP concentrations, without detecting any transition from multisite to unisite rates.…”

Section: Other Ligandsmentioning

confidence: 63%

Modulation of the H+/ATP coupling ratio by ADP and ATP as a possible regulatory feature in the F-type ATP synthases

Turina

2022

Front. Mol. Biosci.

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F-type ATP synthases are transmembrane enzymes, which play a central role in the metabolism of all aerobic and photosynthetic cells and organisms, being the major source of their ATP synthesis. Catalysis occurs via a rotary mechanism, in which the free energy of a transmembrane electrochemical ion gradient is converted into the free energy of ATP phosphorylation from ADP and Pi, and vice versa. An ADP, tightly bound to one of the three catalytic sites on the stator head, is associated with catalysis inhibition, which is relieved by the transmembrane proton gradient and by ATP. By preventing wasteful ATP hydrolysis in times of low osmotic energy and low ATP/ADP ratio, such inhibition constitutes a classical regulatory feedback effect, likely to be an integral component of in vivo regulation. The present miniview focuses on an additional putative regulatory phenomenon, which has drawn so far little attention, consisting in a substrate-induced tuning of the H+/ATP coupling ratio during catalysis, which might represent an additional key to energy homeostasis in the cell. Experimental pieces of evidence in support of such a phenomenon are reviewed.

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Evidence for functional heterogeneity among the catalytic sites of the bovine heart mitochondrial F1-ATPase.

Cited by 58 publications

References 42 publications

The α₃(βY₃₄₁W)₃γ Subcomplex of the F₁-ATPase from the Thermophilic Bacillus PS3 Fails to Dissociate ADP When MgATP Is Hydrolyzed at a Single Catalytic Site and Attains Maximal Velocity When Three Catalytic Sites Are Saturated with MgATP^†

The α₃(βY₃₄₁W)₃γ Subcomplex of the F₁-ATPase from the Thermophilic Bacillus PS3 Fails to Dissociate ADP When MgATP Is Hydrolyzed at a Single Catalytic Site and Attains Maximal Velocity When Three Catalytic Sites Are Saturated with MgATP^†

Catalytic sites of Escherichia coli F1-ATPase. Characterization of unisite catalysis at varied pH

Modulation of the H+/ATP coupling ratio by ADP and ATP as a possible regulatory feature in the F-type ATP synthases

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Evidence for functional heterogeneity among the catalytic sites of the bovine heart mitochondrial F1-ATPase.

Cited by 58 publications

References 42 publications

The α3(βY341W)3γ Subcomplex of the F1-ATPase from the Thermophilic Bacillus PS3 Fails to Dissociate ADP When MgATP Is Hydrolyzed at a Single Catalytic Site and Attains Maximal Velocity When Three Catalytic Sites Are Saturated with MgATP†

The α3(βY341W)3γ Subcomplex of the F1-ATPase from the Thermophilic Bacillus PS3 Fails to Dissociate ADP When MgATP Is Hydrolyzed at a Single Catalytic Site and Attains Maximal Velocity When Three Catalytic Sites Are Saturated with MgATP†

Catalytic sites of Escherichia coli F1-ATPase. Characterization of unisite catalysis at varied pH

Modulation of the H+/ATP coupling ratio by ADP and ATP as a possible regulatory feature in the F-type ATP synthases

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The α₃(βY₃₄₁W)₃γ Subcomplex of the F₁-ATPase from the Thermophilic Bacillus PS3 Fails to Dissociate ADP When MgATP Is Hydrolyzed at a Single Catalytic Site and Attains Maximal Velocity When Three Catalytic Sites Are Saturated with MgATP^†

The α₃(βY₃₄₁W)₃γ Subcomplex of the F₁-ATPase from the Thermophilic Bacillus PS3 Fails to Dissociate ADP When MgATP Is Hydrolyzed at a Single Catalytic Site and Attains Maximal Velocity When Three Catalytic Sites Are Saturated with MgATP^†