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Evaluation of equilibrium constants by affinity chromatography.

ABSTRACT: Theoretical expressions are derived for affinity chromatography of systems comprising an acceptor A with one binding site for attachment to a functional group X on the column matrix and one site for interaction with a small ligand B that specifically affects its elution. From a general relationship covering all possible interactions between A, B and X simpler expressions are derived for affinity systems in which only two equilibria operate. Methods are suggested whereby these simpler systems may be characterized in terms of the two pertinent equilibrium constants and the concentration of matrix-bound constituent. The means by which the theory may be adapted to affinity chromatography of acceptors with multiple binding sites for ligand is also illustrated. Results of partition experiments on the Sephadex G-100-lysozyme-d-glucose system in acetate-chloride buffer (I=0.17m), pH5.4, are used to demonstrate the feasibility of evaluating quantitatively affinity-chromatography interactions. Values of 30m(-1) and 1.2x10(6)m(-1) are obtained for the equilibrium constants for the reactions of lysozyme with glucose and Sephadex respectively, there being only an occasional binding site in the polysaccharide matrix (approximately 1 in 10(5) glucose residues). In a second experimental study the phytohaemagglutinin from Ricinus communis is subjected to frontal chromatography on Sepharose 4B in the presence of different concentrations of d-galactose, the results illustrating some of the difficulties and limitations that are likely to be encountered in quantitative studies of affinity-chromatographic systems.

SUBMITTER: Nichol LW

PROVIDER: S-EPMC1168400 | biostudies-other | 1974 Nov

REPOSITORIES: biostudies-other

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Project description:The caged complex between orotidine 5'-monophosphate decarboxylase (ScOMPDC) and 5-fluoroorotidine 5'-monophosphate (FOMP) undergoes decarboxylation ?300 times faster than the caged complex between ScOMPDC and the physiological substrate, orotidine 5'-monophosphate (OMP). Consequently, the enzyme conformational changes required to lock FOMP at a protein cage and release product 5-fluorouridine 5'-monophosphate (FUMP) are kinetically significant steps. The caged form of ScOMPDC is stabilized by interactions between the side chains from Gln215, Tyr217, and Arg235 and the substrate phosphodianion. The control of these interactions over the barrier to the binding of FOMP and the release of FUMP was probed by determining the effect of all combinations of single, double, and triple Q215A, Y217F, and R235A mutations on kcat/Km and kcat for turnover of FOMP by wild-type ScOMPDC; its values are limited by the rates of substrate binding and product release, respectively. The Q215A and Y217F mutations each result in an increase in kcat and a decrease in kcat/Km, due to a weakening of the protein-phosphodianion interactions that favor fast product release and slow substrate binding. The Q215A/R235A mutation causes a large decrease in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of OMP, which are limited by the rate of the decarboxylation step, but much smaller decreases in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of FOMP, which are limited by the rate of enzyme conformational changes. By contrast, the Y217A mutation results in large decreases in kcat/Km for ScOMPDC-catalyzed decarboxylation of both OMP and FOMP, because of the comparable effects of this mutation on rate-determining decarboxylation of enzyme-bound OMP and on the rate-determining enzyme conformational change for decarboxylation of FOMP. We propose that kcat = 8.2 s(-1) for decarboxylation of FOMP by the Y217A mutant is equal to the rate constant for cage formation from the complex between FOMP and the open enzyme, that the tyrosyl phenol group stabilizes the closed form of ScOMPDC by hydrogen bonding to the substrate phosphodianion, and that the phenyl group of Y217 and F217 facilitates formation of the transition state for the rate-limiting conformational change. An analysis of kinetic data for mutant enzyme-catalyzed decarboxylation of OMP and FOMP provides estimates for the rate and equilibrium constants for the conformational change that traps FOMP at the enzyme active site.

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Evaluation of equilibrium constants by affinity chromatography.

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