Cooperativity among manganese-binding sites in the H+-ATPase of chloroplasts.
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Abstract:
Coupling factor, isolated from lettuce chloroplasts, contained several binding sites for Mn2+ ions.Three of these sites showed strong cooperative interactions having a Hill coefficient of 2.9 f 0.20 and a Kd of 14.7 f 0.44 p ~. Three additional non-interacting Mn2+-binding sites were found with a Kd of 46.7 f 2.3 p ~.Chemical modification with naphthylglyoxal of 1 arginyl residuelchloroplast coupling factor 1, which inhibited ATPase activity, inhibited the cooperativity among the sites but did not prevent Mn2+ binding to the enzyme.It is suggested that the cooperative interaction among the Mn2+-binding sites is an expression of the interaction among the active sites of the enzyme which is required for catalysis.Keywords:
Cooperativity
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F1-ATPase catalyses ATP hydrolysis and converts the cellular chemical energy into mechanical rotation. The hydrolysis reaction in F1-ATPase does not follow the widely believed Michaelis-Menten mechanism. Instead, the hydrolysis mechanism behaves in an ATP-dependent manner. We develop a model for enzyme kinetics and hydrolysis cooperativity of F1-ATPase which involves the binding-state changes to the coupling catalytic reactions. The quantitative analysis and modeling suggest the existence of complex cooperative hydrolysis between three different catalysis sites of F1-ATPase. This complexity may be taken into account to resolve the arguments on the bindingchange mechanism in F1-ATPase.
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We investigated the mechanism by which 9‐ethynylphenanthrene (9EP) inactivates cytochrome P450 2B4 (CYP2B4). Our results demonstrated that 9EP is a potent mechanism‐based inactivator (MBI) of CYP2B4 with a partition ratio and k inact of 0.2 and 0.25 min −1 , respectively. More importantly the kinetics of the MBI exhibit homotropic cooperativity with a Hill coefficient of 2.5 and S 50 of 4.5 μM. To the best of our knowledge, this is the first report of homotropic cooperativity in the mechanism‐based inactivation of P450s. A fully modified CYP2B4 was purified to homogeneity, and its structure was solved by X‐ray crystallography. Based on this crystal structure, 9EP is covalently attached to the Oγ of Thr 302 via an ester bond, resulting in inward rotation of Phe 297 and Phe 206. It is unlikely that the active site of CYP2B4 can accommodate two 9EP molecules. To explore other binding site(s) responsible for cooperativity, fluorescence quenching resulting from 9EP binding to CYP2B4 was investigated. These studies revealed two distinct binding sites for 9EP in CYP2B4. A high affinity site with a K d of ~46 nM was observed in the modified CYP2B4, which likely arises from the binding of 9EP to a peripheral site. In contrast, a high affinity site as well as a low affinity site were associated with the binding of 9EP to the unmodified CYP2B4. These results suggest that 9EP binds to two distinct sites in CYP2B4 that function cooperatively.
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Ligands change the chemical and mechanical properties of polymers. In particular, single strand binding protein (SSB) non-specifically bounds to single-stranded DNA (ssDNA), modifying the ssDNA stiffness and the DNA replication rate, as recently measured with single-molecule techniques. SSB is a large ligand presenting cooperativity in some of its binding modes. We aim to develop an accurate kinetic model for the cooperative binding kinetics of large ligands. Cooperativity accounts for the changes in the affinity of a ligand to the polymer due to the presence of another bound ligand. Large ligands, attaching to several binding sites, require a detailed counting of the available binding possibilities. This counting has been done by McGhee and von Hippel to obtain the equilibrium state of the ligands-polymer complex. The same procedure allows to obtain the kinetic equations for the cooperative binding of ligands to long polymers, for all ligand sizes. Here, we also derive approximate cooperative kinetic equations in the large ligand limit, at the leading and next-to-leading orders. We found cooperativity is negligible at the leading-order, and appears at the next-to-leading order. Positive cooperativity (increased affinity) can be originated by increased binding affinity or by decreased release affinity, implying different kinetics. Nevertheless, the equilibrium state is independent of the origin of cooperativity and only depends on the overall increase in affinity. Next-to-leading approximation is found to be accurate, particularly for small cooperativity. These results allow to understand and characterize relevant ligand binding processes, as the binding kinetics of SSB to ssDNA, which has been reported to affect the DNA replication rate for several SSB-polymerase pairs.
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The synthesis of a heteroditopic receptor which exhibits positive cooperativity for the binding of phosphate ion pairs under physiological conditions. Optimised complementarity between crown ether host and metal guest leads to increased binding affinity, Ka.
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Complementarity (molecular biology)
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The macrobicyclic polyether (1) is the first "small" model system which exhibits a binding cooperativity. It could be shown by 600-MHz 1H-NMR studies that binding of one Hg(CN)2 to (1) increases its binding ability for a second Hg(CN)2 by a factor of ten.
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Allosteric enzyme
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Binding events involving the reversible association of ligands with polymeric lattices of binding sites are common in biology and frequently exhibit significant cooperativity in binding. Positive and negative cooperativity in binding may be detected by characteristic changes in binding curves for multiple binding, compared to the binding expected for simple, independent binding events that are based on combinatorial considerations only. Cooperativity arises from ligand‐dependent interactions distinct from binding per se. Ligand‐dependent nearest neighbor interactions may be of two types referred to as ligand‐lattice (which can only occur if a bound ligand is unneighbored) and ligand‐ligand (which can occur if two or more bound ligands are adjacent). The molecular mechanisms underlying these two sources of cooperativity are not the same. Identical cooperative binding curves may be produced by changes from unity in parameters representing either one or both of these interaction types. Positive cooperativity may equally result from destabilizing ligand‐lattice interactions that disfavor initial, unneighbored binding, stabilizing ligand‐ligand interactions that favor subsequent, neighbored binding, or both. The structural origins of these are different, and cooperativity may emerge from multiple structural interactions.
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