THE STATES OF TYROSYL RESIDUES IN THERMOLYSIN AS EXAMINED BY NITRATION AND PH-DEPENDENT IONIZATION

The states of 28 tyrosyl residues of thermolysin have been characteriz ed by means of pH-jump studies and nitration with tetranitromethane. T he ionization states of phenolic groups of the tyrosyl residues have a lso been estimated by spectrophotometric titration of the absorption c hange at 295 nm. The ionization of 16 tyrosyl residues was completed w ithin 15 s after a pH-jump, and these residues are considered to be lo cated on the surface of thermolysin. On the other hand, the ionization of the other 12 residues required 15 s to 10 min, suggesting the occu rrence of a conformational change which leads to the exposure of the b uried tyrosyl residues to the solvent. Sixteen tyrosyl residues were n itrated and categorized into three classes according to reactivity. Th e second-order rate constants of the respective classes of tyrosyl res idues for nitration were evaluated as 3.32, 0.52, and 0.18 M(-1). min( -1), and their apparent pK(a) values were estimated to be 10.2, 11.4, and 11.8. Tyrosyl residues in the first class were considered to be lo cated almost freely on the surface, while those in the second and thir d classes might be in constrained states.