Occurrence and Characteristics of a Rapid Exchange of Phosphate Oxygens Catalyzed by Sarcoplasmic Reticulum Vesicles

Kanazawa, Tohru; Boyer, Paul D.

doi:10.1016/s0021-9258(19)44022-2

Cited by 206 publications

(44 citation statements)

References 32 publications

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“…The concentrations of free Ca2+ were calculated by using an apparent dissociation constant of 3.9 X 10~7 M for Ca2+-EGTA with the computer program of Fabiato (1988) Ko.5 = 2.1 µ cytoplasmic Ca2+ with empty SRV (O), K0.5 = 4 µ cytoplasmic Ca2+ with loaded SRV (•), and a Hill coefficient of « = 2, which corresponds to the binding of two Ca2+ ions to the cytoplasmic sites with positive cooperativity. It is well-known that cytoplasmic Ca2+ inhibits phosphorylation of the enzyme by P¡ at equilibrium (Kanazawa & Boyer, 1973) by binding to the high-affinity cytoplasmic sites, as shown in Scheme 3. The concentration of cytoplasmic saturated with 40 mM lumenal Ca2+.…”

Section: Resultsmentioning

confidence: 99%

“…This result shows that binding of Ca.2* to the lumenal sites of the unphosphorylated enzyme does not cause a significant change in the rate constant of kjng = 50 s_1 for dissociation of Mg2+ from the catalytic site. This is in contrast to the strong interaction between the cytoplasmic Ca2+-binding sites and the catalytic site: (1) Phosphorylation of the enzyme by ATP occurs when the cytoplasmic sites are occupied by Ca2+, while phosphorylation of the enzyme by P¡ occurs when the cytoplasmic sites are free from Ca2+ (Yamamoto & Tonomura, 1967;Makinose, 1969;Kanazawa & Boyer, 1973; Pickart & Jencks, 1984). (2) Mg2+ dissociates from the catalytic site with Ka = 8.7 mM in the absence of Ca2+ (Punzengruber et al, 1978), which is ~10 times weaker than Ka = 0.94 mM from the enzyme with Ca2+ bound at the cytoplasmic sites (Reinstein & Jencks, 1993).…”

Section: Resultsmentioning

confidence: 99%

“…This is in contrast with the strong interaction that exists between the cytoplasmic high-affinity sites for Ca2+ and the catalytic site, although these sites are separated by at least 30 Á (Highsmith & Murphy, 1984;Scott, 1985;Toyoshima et al, 1993). The enzyme is phosphorylated only by ATP when cytoplasmic Ca2+ is bound to the high-affinity sites, whereas it is phosphorylated only by P, in the absence of cytoplasmic Ca2+ (Yamamoto & Tonomura, 1967; Makinose, 1969; Kanazawa & Boyer, 1973;Pickart & Jencks, 1984). The dissociation constant of Mg2+ from the catalytic site is decreased by ~10-fold, from 8.7 mM (Punzengruber et al, 1978) to 0.94 mM (Reinstein & Jencks, 1993), when cytoplasmic Ca2+ is bound to the high-affinity sites.…”

Section: Discussionmentioning

confidence: 99%

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Lumenal and Cytoplasmic Binding Sites for Calcium on the Calcium ATPase of Sarcoplasmic Reticulum Are Different and Independent

Myung

Jencks²

1994

Biochemistry

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The calcium ATPase of sarcoplasmic reticulum reacts with inorganic phosphate (Pi) to form phosphoenzyme that can bind two Ca2+ ions from the lumen of intact vesicles. Therefore, as the concentration of lumenal Ca2+ is increased, the concentration of phosphoenzyme at equilibrium increases; however, it levels off at lower maximal concentrations with decreasing concentrations of Pi. This requires that two Ca2+ ions can bind to lumenal binding sites of both the phosphoenzyme and the unphosphorylated enzyme. If lumenal Ca2+ could bind only to the phosphoenzyme, saturating concentrations of lumenal Ca2+ would drive phosphoenzyme formation to completion even at low concentrations of Pi. Phosphorylation is inhibited by cytoplasmic Ca2+ with K0.5 = 2.1 and 4 microM in the absence and in the presence of 40 mM lumenal Ca2+, respectively. K0.5 = 4 microM is much lower than K0.5 = 70 microM, which is expected if lumenal Ca2+ could bind only to the phosphoenzyme. Occupancy of the lumenal sites on the unphosphorylated enzyme by Ca2+ does not significantly change the rate constants of kp = 220 s-1 for phosphorylation by ATP, kCa = 90 s-1 for dissociation of Ca2+, and kMg = 50 s-1 for dissociation of Mg2+. We conclude that the calcium ATPase has two low-affinity lumenal Ca(2+)-binding sites that are independent of the high-affinity cytoplasmic Ca(2+)-binding sites. The results are consistent with a mechanism of Ca2+ transport in which phosphorylation of the enzyme by ATP drives the translocation of two Ca2+ ions from the high-affinity to the low-affinity sites.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Lumenal and Cytoplasmic Binding Sites for Calcium on the Calcium ATPase of Sarcoplasmic Reticulum Are Different and Independent

Myung

Jencks²

1994

Biochemistry

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“…This is in contrast to the strong inhibition by cytoplasmic Ca2+ of phosphorylation of the enzyme by P¡ (Kanazawa & Boyer, 1973). It is well established that a strong interaction exists between the high-affinity cytoplasmic sites for Ca2+ and the catalytic site, although these sites are separated by at least 30 A (Highsmith & Murphy, 1984;Scott, 1985;Toyoshima et al, 1993).…”

Section: Discussionmentioning

confidence: 87%

“…It is well established that a strong interaction exists between the high-affinity cytoplasmic sites for Ca2+ and the catalytic site, although these sites are separated by at least 30 A (Highsmith & Murphy, 1984;Scott, 1985;Toyoshima et al, 1993). The enzyme is phosphorylated by ATP only when cytoplasmic Ca2+ is bound to the highaffinity cytoplasmic sites, whereas it is phosphorylated by Pi only in the absence of cytoplasmic Ca2+ (Yamamoto & Tonomura, 1967;Makinose, 1969;Kanazawa & Boyer, 1973;Pickart & Jencks, 1984). Chaloub et al (1979) have reported that phosphorylation of the enzyme by P¡ at pH 6.2 in the absence of KC1 proceeds with rate constants of 30 s-1 with empty SRV and 2 s-1 with SRV that were actively loaded with Ca2+ in the presence of ATP, and concluded that lumenal Ca2+ inhibits the rate of phosphorylation of the enzyme by P¡.…”

Section: Discussionmentioning

confidence: 99%

There Is Only One Phosphoenzyme Intermediate with Bound Calcium on the Reaction Pathway of the Sarcoplasmic Reticulum Calcium ATPase

Myung¹,

Jencks²

1995

Biochemistry

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Identical first-order rate constants for phosphorylation of the calcium ATPase of sarcoplasmic reticulum by bound inorganic phosphate (Pi) of 25 +/- 2 s-1 with empty vesicles and 25 +/- 1 s-1 with vesicles that were passively loaded with 40 mM Ca2+ were obtained by treating the reaction as an approach to equilibrium (4 mM [32P]Pi, 20 mM MgCl2, 10 mM EGTA, and 100 mM KCl at pH 7.0 and 25 degrees C). The formation of ADP-sensitive phosphoenzyme from Pi with Ca(2+)-loaded vesicles also proceeds with a first-order rate constant of 25 s-1 and no detectable induction period. These identical rate constants show that lumenal Ca2+ does not inhibit the rate of phosphorylation of the enzyme by bound Pi and that there is no significant kinetic barrier for the conformational change that converts an ADP-insensitive to an ADP-sensitive phosphoenzyme intermediate with bound Ca2+. We conclude that there is no evidence for the existence of two stable phosphoenzyme intermediates with bound Ca2+, such as E1 approximately P.Mg.Ca2 and Ca2.E2-P.Mg, that are included in the E1-E2 and related two-state models for calcium transport by this enzyme. In general, coupling of a physical reaction, such as muscle contraction or vectorial transport, to a chemical reaction, such as ATP hydrolysis, requires more than two states in the reaction cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

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Energie, Leben und ATP (Nobel-Vortrag)

Boyer

1998

Angewandte Chemie

Self Cite

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Die Zeit genutzt hat Paul Boyer während eines Vortrags, der ihn nicht sonderlich interessierte: Beim Nachdenken über die verwirrenden Ergebnisse von 18O‐Austauschexperimenten erkannte er, daß nicht die Synthese von ATP, sondern hauptsächlich dessen anschließende Freisetzung von der protonenmotorischen Kraft abhängt, die aus den Oxidationsprozessen der Atmungskette stammt – das Konzept des Bindungswechsel‐Mechanismus der ATP‐Synthese war geboren. Für die ATP‐Synthase, die die Bildung von ATP aus ADP und anorganischem Phosphat, eine der wichtigsten Reaktionen in der Natur, katalysiert, fordert dieses Konzept Eigenschaften wie sequentielle Konformationsänderungen und eine diese Änderungen bewirkende Rotation, die dieses Enzym als einzigartige molekulare Maschine erscheinen lassen.

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Occurrence and Characteristics of a Rapid Exchange of Phosphate Oxygens Catalyzed by Sarcoplasmic Reticulum Vesicles

Cited by 206 publications

References 32 publications

Lumenal and Cytoplasmic Binding Sites for Calcium on the Calcium ATPase of Sarcoplasmic Reticulum Are Different and Independent

Lumenal and Cytoplasmic Binding Sites for Calcium on the Calcium ATPase of Sarcoplasmic Reticulum Are Different and Independent

There Is Only One Phosphoenzyme Intermediate with Bound Calcium on the Reaction Pathway of the Sarcoplasmic Reticulum Calcium ATPase

Energie, Leben und ATP (Nobel-Vortrag)

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