Studies on thermolysin. I. Effects of chemical modifications on the activity of thermolysin.
1. When thermolysin was treated with a 100-fold molar excess of 2,4,6-trinitrobenzene-1-sulfonate at pH 8.0 and 37 degrees for 7 h, all 12 amino groups in the enzyme were almost completely trinitrophenylated. The fully trinitrophenylated enzyme still retained more than 80% of its original activity. The amino groups are therefore not essential for activity. 2. When treated with a 100- to 1,000-fold molar excess of N-acetylimidazole at pH 6.5 and 23 degrees for 2 h, thermolysin lost about 54% of its activity with concomitant acetylation of 21 tyrosine residues out of the total of 28 residues. The reaction did not easily proceed any further. This partially inactivated enzyme regained almost full activity upon treatment with hydroxylamine. These modified tyrosine residues are therefore not directly involved in the active site. 3. Thermolysin was not inactivated by treatment with about 100- to 150-fold molar excess of 2-hydroxy-5-nitrobenzyl bromide or dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide at pH 6.0 and room temperature for 1 h both in the presence and absence of 8 M urea. Thus the three tryptophan residues in the enzyme are not accessible to these reagents. When treated with a 4.3- to 43-fold molar excess of N-bromosuccinimide over tryptophan, the enzyme was inactivated to varying extents, depending on the reaction conditions used. In this case, the tyrosine residues appeared to be the most rapidly modified, but tryptophan and histidine residues were also modified with extensive inactivation at higher concentrations of the reagent. The presence of 8 M urea retarded the inactivation.
D000596:Amino Acids / Q000032:analysis; D001974:Bromosuccinimide / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013820:Thermolysin / Q000378:metabolism*; D014302:Trinitrobenzenesulfonic Acid / Q000494:pharmacology; D014443:Tyrosine; D014508:Urea / Q000494:pharmacology
D000596:Amino Acids; D007093:Imidazoles; D014443:Tyrosine; D014302:Trinitrobenzenesulfonic Acid; D014508:Urea; D013820:Thermolysin; D001974:Bromosuccinimide