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 characterized by means of pH-jump studies and nitration with tetranitromethane. The ionization states of phenolic groups of the tyrosyl residues have also been estimated by spectrophotometric titration of the absorption change at 295 nm. The ionization of 16 tyrosyl residues was completed within 15 s after a pH-jump, and these residues are considered to be located on the surface of thermolysin. On the other hand, the ionization of the other 12 residues required 15 s to 10 min, suggesting the occurrence of a conformational change which leads to the exposure of the buried tyrosyl residues to the solvent. Sixteen tyrosyl residues were nitrated and categorized into three classes according to reactivity. The second-order rate constants of the respective classes of tyrosyl residues 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 located almost freely on the surface, while those in the second and third classes might be in constrained states.