Allosteric Effects: Binding Cooperativity in a Subunit Model
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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.Keywords:
Cooperativity
Cooperative binding
Allosteric enzyme
Cooperativity
Allosteric enzyme
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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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Abstract Magnitude of cooperativity of a hypothetical m ‐subunit allosteric system of hemoglobin type is discussed, based on computation of the amount of O 2 to be transferred to the corresponding monomeric nonallosteric system of myoglobin type. For quantitative discussion, “allosteric gain” is defined as the difference between the amount of O 2 to be transferred from the allosteric to the nonallosteric system and that from the hypothetical system of average O 2 affinity to the nonallosteric system. These two quantities, allosteric gain and amount of O 2 transfer, are quite sensitive to equilibrium constant ratio, especially K m /K 1 , ( K i+1 / K i ) max , and i. Hill's coefficient appropriately describes neither the amount of O 2 transferred nor allosteric gain. Allosteric gain or the amount of O 2 transfer which is best characterized by the total cooperativity index ( K m /K 1 ) and the local cooperativity indices ( K i+1 /K i ) seem to be appropriate measures of the magnitude of cooperativity.
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Cooperative binding
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Structural Biology
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Kinetic studies of L-Alanine dehydrogenase from Bacillus subtilis-catalyzed reactions in the presence of were carried out. The substrate (L-alanine) saturation curve is hyperbolic in the absence of the metal ion but it becomes sigmoidal when is added to the reaction mixture indicating the positive cooperative binding of the substrate in the presence of zinc ion. The cooperativity of substrate binding depends on the xinc ion concentration: the Hill coefficients () varied from 1.0 to 1.95 when the zinc ion concentration varied from 0 to . The inhibition of AlaDH by is reversible and noncompetitive with respect to (). itself binds to AlaDH with positive cooperativity and the cooperativity is independent of substrate concentration. The Hill coefficients of substrate biding in the presence of are not affected by the enzyme concentration indicating that binding does not change the polymerization-depolymerization equilibria of the enzyme. Among other metal ions, appears to be a specific reversible inhibitor inducing conformational change through the intersubunit interaction. These results indicate that is an allosteric competitive inhibitor and substrate being a non-cooperative per se, excludes the from its binding site and thus exhibits positive cooperativity. The allosteric mechanism of AlaDh from Bacillus subtilis is consistent with both MWC and Koshland's allosteric model.
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Saturation vapor curve
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Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring.
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Cooperativity
Cooperative binding
Allosteric enzyme
Protein Dynamics
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Abstract Cells can respond to changes in their environment by altering the flow through particular metabolic pathways. Such a change in any metabolic step is due to certain key enzymes that have the ability to alter their rate of activity. Such enzymes are defined as allosteric. The extent to which these enzymes adopt either the active conformation or the inactive conformation depends on their response to appropriate positive or negative signals. An easily measured feature of allosteric enzymes is the cooperativity that they show in a kinetic experiment.
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Metabolic pathway
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Abstract The effects of activator molecule and repressive molecule on binding process between allosteric enzyme and substrate are discussed by considering the heterotropic effect of the regulating molecule that binds to allosteric enzyme. A model of allosteric enzyme with heterotropic effect is presented. The cooperativity and anticooperativity in the regulation process are studied.
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Cooperative binding
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