KINETICS AND THERMODYNAMICS OF LIGAND BINDING TO SPERM WHALE MYOGLOBIN

Abstract

Association rate constants, dissociation rate constants, and quantum yields for the binding of CO, O(,2), and 11 alkyl isocyanides to protocheme mono-3-(1-imidazoyl)propylamide monomethyl ester (model heme) were measured in benzene and myristyltrimethylammonium bromide micelles. The same kinetic parameters were determined for these ligands binding to sperm whale myoglobin. The protein rate constants were determined as a function of glycerol concentration and temperature. The results were analyzed in terms of a simple, single kinetic barrier model and a more detailed three step binding scheme. Model heme allows an assessment of the influence of the protein structure on the rates and extents of ligand binding. When model heme is in an isotropic environment, as in benzene, the association rate constants (k') for isonitrile (RNC) binding are all about 2 x 10('8) M('-1)sec('-1) and roughly independent of the size and structure of the alkyl group. When model heme is dissolved in an aqueous soap suspension, k' for RNC binding increases monotonically as the surface area of the alkyl side chain increases. The ligands are partitioning between the aqueous and micellar phases. In the highly anisotropic environment of myoglobin, RNC binding exhibits a very complex dependence on ligand size and stereochemistry. Increasing solvent viscosity, by adding glycerol, decreases k' for CO binding to free protoheme. In contrast, k' for binding to myoglobin is either unchanged (RNC) or increases (CO) with increasing glycerol concentration. Solvent viscosity does not attenuate the observed protein association rates. However, the association equilibrium constants (K(,A)) for myoglobin are increased 2 to 4-fold in going from 0 to 75% glycerol. Two mechanisms for this effect are identified. For CO and O(,2), a four-fold decrease in gas solubility is correlated with the four-fold K(,A) increase with increasing glycerol concentration. For RNCs, preferential stabilization of the bound RNC-myoglobin complex accounts for the observed K(,A) increases. The activation energies for CO and O(,2) association with myoglobin are similar and relatively small ((DBLTURN)4-5 kcal/mol), while RNCs exhibit larger values that depend on both the size and structure of the alkyl group. The (DELTA)H(DEGREES) and (DELTA)S(DEGREES) for O(,2) binding are -13.5 kcal/mol and -20 eu. The entropy change suggests that O(,2) orders the protein, possibly by forming a hydrogen bond to the distal histidine. In contrast, the corresponding CO values are -9.6 kcal/mol and +0.8 eu indicating, that other than bond formation, CO has little impact on the protein. For RNCs, (DELTA)H(DEGREES) and (DELTA)S(DEGREES) exhibit a complex dependence on ligand size and structure which can be rationalized with the steric constraints of the active site

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oai:scholarship.rice.edu:1911/16023Last time updated on 6/11/2012

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