Reaction cycle pumps

Fig. 4. E1-E2 reaction cycle of the Na,K-pump with four major occluded conformations and ping-pong sequential cation translocation. The phosphoforms can occlude Na" and dephosphoforms can occlude or Rb. Na and K without brackets are cations bound to an open form such that they can exchange with medium cations [Na ] or [K ] within brackets are occluded and prevented from exchanging with medium cations. It is proposed that release of Na cxt accompanies transition from EiP[3Na] to E2P[2Na], since the capacity for occlusion of Na in the ouabain-stabilized E2P form is lower than in the EjP form prepared by incubation with CrATP [29] or oligomycin [89].

The CP MAS NMR spectroscopy has been also extensively used for studies of proteins containing retinylidene chromophore like proteorhodopsin or bacteriorhodopsin. Bacteriorhodopsin is a protein component of purple membrane of Halobacterium salinarium.71 7 This protein contains 248 amino acids residues, forming a 7-helix bundle and a retinal chromophore covalently bound to Lys-216 via a Schiff base linkage. It is a light-driven proton pump that translocates protons from the inside to the outside of the cell. After photoisomerization of retinal, the reaction cycle is described by several intermediate states (J, K, L, M, N, O). Between L and M intermediate states, a proton transfer takes place from the protonated Schiff base to the anionic Asp85 at the central part of the protein. In the M and/or N intermediate states, the global conformational changes of the protein backbone take place. 

In both schemes, the specificities of the pump for catalysis change in the two enzyme states. Jencks points out that coupling is determined (a) by the chemical specificity achieved in catalyzing phosphoryl transfer to and from the enzyme (wherein E-Ca2 reversibly binds ATP, and E reacts reversibly with orthophosphate), and (b) by the vectorial specificity for ion binding and dissociation (wherein E reversibly binds/dissociates cytoplasmic calcium ion, and E—P reversibly binds/dissociates luminal calcium). There must be a single conformation change during the reaction cycle between Ei and E2 in the free enzyme and from Ei P-Ca2 to E2-P-Ca2 after enzyme phosphorylation. 

Figure 3. Schematic representation of the reaction cycle of SERCA pumps. The SERCA pumps exist in two conformational state El binds Ca2+ with high affinity at the cytoplasmic site of the SER membrane, while E2 has low affinity for Ca2+ and thus releases it on the opposite site of the membrane. ATP phosphorylates a highly conserved aspartic acid residue allowing for the translocation of Ca2+ in the SER lumen...

Silica gels were deuterated by pumping D20 at equilibrium vapour pressure over the sample. Time and temperature of both the adsorption and evacuation step were varied. The continuous flow of D20 vapour overcomes the need for repeating the procedure in several reaction cycles. The spectrum of silica gel, pretreated at 473 K under vacuum, before and after room temperature deuteration is shown in figure 3.5. Optimal exchange was observed if the adsorption was performed for 1 h at room temperature and the evacuation at the same temperature as pretreatment. 

Tanford, C. (1984). Twenty questions concerning the reaction cycle of the sarcoplasmic reticulum calcium pump. Crit. Rev. Biochem. 17, 123-151. 

Figure 4. Simplified scheme for the reaction cycle in Ca2+ pumps. The pumps may adopt two major conformations E, and E2. The E, conformation shows high affinity for two Ca2+ (SERCA pumps) or one Ca2+ (PMCA pumps) on the cis side. Ca2+ binding greatly enhances the pumps ATPase activity, leading to the rapid formation of the h igh-energy phosphorylated intermediate E, P and occlusion (occ) of the transported Ca2+ ion(s). Ca2+ translocation across the membrane presumably occurs concomitantly with the release of energy stored as conformational constraint during the transition from the E, P to the low-energy E2-P conformation. Ca2+ affinity on the trans side is low and Ca2+ is therefore released. This is followed by hydrolysis of the phosphoenzyme and a poorly understood rearrangement step(s) from the E2 to the E, conformation.

Figure 8 shows a scheme of the reaction cycle of copper ATPases, assuming that they work by a mechanism analogous to that of Ca - or Na+, K+-ATPases. To pump ions, the enzyme must cycle between a state with a high-affinity copper-binding site accessible from only one side of the membrane and a low-affinity state in which the copper cavity is accessible from the other side of the membrane. The high- and low-affinity forms of P-type ATPases were initially named Ei and E2 by Racker (1980) and for many years these ATPases were called E]E2-ATPases, until they were renamed P-type by Pedersen and Carafoli (1987a). 

Thirty years of research with bacteriorhodopsin has provided answers to many questions about how protons are transported by transmembrane pumps. In this small seven-transmembrane protein, absorption of light by the retinal chromophore Initiates a reaction cycle in which the initial state recovers through multiple conformational changes of the retinal and the protein, and a proton Is translocated stepwise from one side of the membrane to the other. Spectroscopy, extensive use of site-specific mutations, and crystallography have defined the photocycle reactions in atomic detail and provide a step-by-step description of the proton transfers, the transient local and global perturbations in the protein and how they arise, and the energy flow through the system, which add up to the mechanism of the pump. 

FIGURE 11.5 The reaction cycle of Ca -ATPase pumps. In the Ei conformation of the pump, is bound with high affinity at the cytoplasmic side of the plasma membrane. In the E2 configuration, the binding site exposes Ca to the external site of the plasma membrane, where its lower affinity for Ca favours its release. (Adapted fmm Di Leva et at, 2008. Copyright 2008, with permission fmm Elsevier.)... 

More than 20 crystal structures have been reported for SERCAla in 9 different states that approximately cover the entire reaction cycle. The entire reaction cycle can be described essentially with the 4 principal structures depicted in Figure 11. Ab, including a fairly detailed scenario of ion pumping, a description of how the affinity of the transmembrane Ca -binding sites is altered, and how the luminal gate is opened and closed by events that occur around the phosphorylation site more than 50 A away. 

The reaction mechanism of the SERCA- and PMCA-type Ca2+ pumps is essentially identical, involving the transient formation and hydrolysis of an acylphos-phate bond to an aspartate residue. For more information on the elementary steps of the reaction cycle the reader is referred to the reviews cited in the introduction and references therein. 

The Pm state has a very high midpoint potential and it is readily reduced. Transfer of an electron into the catalytic site in state Pm, provided from cytochrome r—> Cua—> heme a, probably results in reduction of the Tyr288 radical. Electron transfer to the catalytic site is coupled to a series of proton transfers, which is thought to be the same every time an electron is transferred to the catalytic site in the reaction cycle two protons are taken up from the N-side of the protein and one is released from the P-side. One of the protons taken up goes to the catalytic site (substrate proton) and the other proton is pumped. 

In a closed system, an ethereal solution of hydrazoic acid is frozen with liquid nitrogen, and diborane (BP, -92.5°) is condensed in. The mixture is allowed to slowly warm up to room temperature. After melting, the fluid is rapidly stirred. Reaction starts at -20 to - 10°C with gas evolution and continues for about 1 hr. The hydrogen is then pumped off, and the reaction recommences. Reaction and pump cycles are repeated until 95% of the total hydrogen has been removed. 

Compare the sarcoplasmic Ca -ATPase and the Na -K pump in terms of functional sites and reaction cycles. 

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