Chromium(III)ATP inactivating (Na++ K+)‐ATPase supports Na+‐Na+ and Rb+‐Rb+ exchanges in everted red blood cells but not Na+,K+ transport

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The exchange-inert tetra-ammino-chromium complex of ATP [Cr(NH3)4ATP], unlike the analogous cobalt complex CO(NH,)~ATP, inactivated Na + /K + -ATPase slowly by interacting with the high-affinity ATP binding .site. The inactivation proceeded at 37°C with an inactivation rate constant of 1.34 x lo-, min-' and with a dissociation constant of 0.62 pM. To assess the potential role of the water ligands of metal in binding and inactivation, a kinetic analysis of the inactivation of Na +/K +-ATPase by Cr(NH,),ATP, and its H20-substituted derivatives Cr(NH3),(H20)ATP, Cr(NH3)2(H20)zATP and Cr(H20)4ATP was carried out. The substitution of the H 2 0 ligands with NH3 ligands increased the apparent binding affinity and decreased the inactivation rate constants of the enzyme by these complexes. Inactivation by Cr(H20),ATP was 29-fold faster than the inactivation by Cr(NH3)4ATP. These results suggested that substitution to Cr(II1) occurs during the inactivation of the enzyme. Additionally hydrogen bonding between water ligands of metal and the enzyme's active-site residues does not seem to play a significant role in the inactivation of Naf/K+-ATPase by Cr(II1)-ATP complexes.Inactivation of the enzyme by Rh(HzO),ATP occurred by binding of this analogue to the high-affinity ATP site with an apparent dissociation constant of 1.8 pM. The observed inactivation rate constant of 2.11 x min-' became higher when Na' or Mg2+ or both were present. The presence of K f however, increased the dissociation constant without altering the inactivation rate constant. High concentrations of Na' reactivated the Rh(HzO),ATP-inactivated enzyme.Co (

Section: Interactionsupporting

confidence: 82%

How do MgATP analogues differentially modify high‐affinity and low‐affinity ATP binding sites of Na⁺/K⁺ ‐ATPase?

Serpersu

Bunk

Schoner

1990

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“…As was found with other MgATP complex analogues [lo, 11,[14][15][16], inactivation of Na+/K+-ATPase by Cr(H,O),AdoPP[CH,]P is hindered by the presence of ATP as well. Fig.…”

Section: Interaction Of Cr(ho)bdopp[ch]p With Na+/k+-atpasementioning

confidence: 76%

Modification of the E₁ATP binding site of Na⁺/K⁺‐ATPase by the chromium complex of adenosine 5′‐[β,γ‐methylene]triphosphate blocks the overall reaction but not the partial activities of the E₂ conformation

Hamer¹,

Schoner²

1993

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The chromium complex of adenosine 5′‐[β,γ‐methylene]triphosphate, Cr(H2O)4AdoPP[CH2]P, inactivates Na+/K+‐ATPase from pig kidney at 37°C with an inactivation velocity constant of 7.1X10‐3min‐1 by binding to the high‐affinity ATP site (E1ATP site). The dissociation constant (Kd) of the analogue at this site is 26 μM, and of ATP 0.8 μM. Inactivation of the overall reaction of Na+/K+‐ATPase by Cr(H2O)4AdoPP[CH2]P did not alter the activities of the E2 conformational state such as K+‐activated p‐nitrophenylphosphatase, 86Rb+ occlusion and [3H]ouabain binding by the ‘backdoor’ phosphorylation. However, [3H]ouabain binding via the forwards reaction from E1ATP in the presence of Na++ Mg2+ is inhibited. K+‐activated p‐nitrophenylphosphatase activity of the Cr(H2O)4AdoPP[CH2]P‐inactivated enzyme decreases when an MgATP analogue, the tetraammine cobalt complex of ATP, Co(NH3)4ATP, is used additionally to inactivate the E2ATP site. The enzyme activity of K+‐activated phosphatase is also lost if the β,γ‐bidentate chromium(III) complex of ATP, Cr(H2O)4ATP, which may form a stable E1‐chromo‐phosphointermediate, is used for the inactivation of Na+/K+‐ATPase. We conclude that the phenomenon of a blockade of the overall reaction of Na+/K+‐ATPase by the formation of a stable E1· CrAdoPP[CH2]P complex, leading thereby to a loss of the partial activities of the E1 conformation, but not of the E2 conformation, is consistent with the postulate of an (αβ)2 diprotomeric nature of the sodium pump. The observation, moreover, that treatment of the sodium pump with Cr(H2O)4ATP but not with Cr(H2O)4AdoPP[CH2]P leads to an inactivation of K+‐activated phosphatase seems to indicate that the formation of a E1‐phosphointermediate affects the E2ATP site.

“…Apparently, eosin is not bound with high affinity to the CrATP-inactivated enzyme. This is expected since CrATP interacts with the highaffinity ATP-binding site of the enzyme in the E, conformation and forms a relatively stable chromium phosphoenzyme complex [6, 28,36,371. Na'/K+-ATPase which was first inactivated to 97% with Co(NH3),ATP and then with CrATP in the presence of 10 mM NaCl and 5 mM MgC12, behaves in a similar manner.…”

Section: Does the Co(nh3j4atp-inactivuted Enzyme Still Have Catalyticmentioning

confidence: 99%

Shift to the Na⁺ from of Na⁺/K⁺‐transporting ATPase due to modification of the low‐affinity ATP‐binding site by Co(NH₃)₄ATP

Scheiner‐Bobis

Esmann

Schoner

1989

Self Cite

1. Inactivation of purified Na+/K'-transporting ATPase by the MgATP complex analogue CO(NH~)~ATP, which binds to the low-affinity ATP-binding site, results in the concomitant inhibition of the K+-activated pnitrophenylphosphatase, which is considered to be a partial reaction catalyzed by the enzyme in the E2 conformational state.2. Complete inactivation of Na+/K+-transporting ATPase by C O ( N H~)~A T P does not alter the ADP/ATP exchange reaction which is considered to be part of the catalytic activity in the El conformation.3. The enzyme binds eosin at the high-affinity ATP-binding site as measured by the change in eosin fluorescence. Eosin binding to the C~(NH~)~ATP-inactivated enzyme is, in contrast to the untreated enzyme, not stimulated by Na+ . Inactivation by C O ( N H~)~A T P increased the half-maximal opposing effect of K + on eosin binding from 1.1 mM in the control to 43.2 mM in the almost completely inactive enzyme. No eosin fluorescence changes were observed when the Co(NH3)4ATP-inactivated enzyme was treated subsequently with CrATP. This MgATP complex analogue forms a stable complex at the high-affinity ATP-binding site. CrATP thus abolishes eosin binding. It is concluded, that Co(NH3)4ATP interacts with Na+/K+-transporting ATPase in the Ez conformationand arrests it there. This affects eosin binding to the high-affinity ATP-binding site, since the K f sensitivity is lost. A possible interpretation of these differing effects of C O ( N H~)~A T P on partial reactions of N a + / K + -transporting ATPase is that the sodium pump works as an diprotomer. It is likely that the arrest of one ct,P protomer in the E2 conformational state by occupancy of the low-affinity ATP-binding site with C O ( N H~)~A T P induces the Na' form (El form) in the corresponding a,p protomer, as is indicated by the unaffected ADP/ATP exchange and the response of the eosin fluorescence on Na' and K f .It is unclear so far, whether the active transport of Na+ and K + through animal cell membranes is catalyzed by the sodium pump [I, 21 in its M,P protomeric or in its (~( , j j )~ diprotomeric form. Reports on the interaction of N a + / K + -transporting ATPase (Na+/K+-ATPase) with 2',3'-0-(2,4,6-trinitrocyclohexadienylidene) adenosine 5'-triphosphate [3, 41, vanadate [5] as well as the recent report on the incorporation of "Na+ and *'Rb+ into the CrATP-inactivated enzyme and its occlusion in the solubilized form [6] favour the concept that the cr,p protomer might fulfil the minimal requirements of the sodium pump as it is described by the socalled Albers-Post model. There are, however, a number of reports which are not consistent with this assumption (for recent reviews see [7 -91). This sometimes rather indirect data supports the concept of a cooperation of a$ protomers and raises the question how the catalytic c( subunits interact [7 -191. We recently showed that the MgATP complex analogue C O ( N H~)~A T P binds to the low-affinity ATP-binding site, which is found in the Ez conformational state [20, 211. In contrast to...