Studies of ligand-mediated conformational changes in enzymes by difference sedimentation velocity in the Optima XL-A ultracentrifuge

Abstract

Difference sedimentation velocity has provided an extremely convenient procedure for detecting and quantifying ligand-mediated conformational changes in enzymes by virture of differences in hydrodynamic volume. However, the replacement of the Beckman model E instrument by the XL-A has necessitated reexamination of the existing method of analysis, which relied upon the comparison of simultaneously recorded distributions of solute in the two cells. After demonstration of the validity of the revised procedure by its application to simulated sedimentation velocity data, differential sedimentation velocity has been used to confirm the effect of phenylalanine on the sedimentation coefficient of rabbit muscle pyruvate kinase. Corresponding studies of the effect of glucose on the sedimentation coefficient of yeast hexokinase have demonstrated the substrate-mediated decrease in enzyme size that is evident from X-ray crystallographic studies, and identified this effect as the consequence of substrate perturbation of a preexisting enzyme isomerization rather than of substrate-induced isomerization of yeast hexokinase.