Studies on Thermolysin

I. Inactivation of Thermolysin by Rose Bengal-catalyzed Photooxidation-Evidence for the Presence of a Critical Histidine Residue

Abstract

1. Thermolysin was rapidly inactivated by rose bengal-catalyzed photooxidation at pH 8.0 and 37°C. The rate of inactivation was pH-dependent and became slower at lower pH values, suggesting the involvement of some histidine residues in the inactivation.
2. Changes in amino acid composition occurred only with some histidine, tryptophan and tyrosine residues. The enzyme appeared to be inactivated almost in parallel with the loss of one or two histidine residues, whereas the rate of loss of tryptophan residue was much slower than that of the enzymatic activity.
3. No significant change in the optical rotatory dispersion curve of the enzyme took place by photooxidation, suggesting the occurrence of little conformational change.
4. These lines of evidence indicate that the photooxidative inactivation of thermolysin is caused mainly by modification of one or two histidine, suggesting the implication of at least one histidine residue in the active center of the enzyme, although the possibility of the presence of tryptophan and tyrosine residues in the active center cannot be excluded.