CONFORMATIONAL STATES OF NATIVE AND DENATURED PHOSPHOGLUCOSE ISOMERASE .I. TITRATION OF EXPOSED AND BURIED AMINO ACID RESIDUES

Rabbit muscle phosphoglucose isomerase, in the native state and under various conditions of denaturation, was subjected to electrometric and spectrophotometric titration with the object of determining the number of exposed and buried histidyl and tyrosyl residues and of investigating their possible role in maintaining the conformation of the enzyme. In native phosphoglucose isomerase, 20 of the total of 47 histidyls and all of the 24 tyrosyls are inaccessible. Acid denaturation normalizes the titration of the 20 anomalous histidyls, and a strongly alkaline medium that of all of the tyrosyls. Sodium dodecyl sulfate (0.5%) allows all of the 12 sulfhydryl groups to be titrated but does not expose any of the tyrosyls, whereas 8 M urea brings all 24 of them into contact with the solvent. The parallel nature of enzyme activity loss and exposure of about 20 histidyls and 20 tyrosyls, on acid or alkaline denaturation, is interpreted in terms of their participation in maintaining the native structure of phosphoglucose isomerase through formation of hydrogen bonds which are stabilized by hydrophobic shielding from competition by water molecules. On the basis of the electrometric titration studies, average intrinsic pK values for the ionization of lysine (10.2) and of imidazole (6.9) and carboxyl groups (4.2) were calculated. In addition, a detailed study of the electrophoretic mobility as a function of the ionic strength yielded an isoelectric point of 8.5 at 1° when extrapolated to zero ionic strength. The isoionic point was found to be 7.47 by direct measurement at 30°. © 1969, American Chemical Society. All rights reserved.