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J. Biochem, 1974, Vol. 76, No. 4 871-878
© 1974 Japanese Biochemical Society

research-article

Changes of Thermostability and Activity of Thermolysin on Nitration with Tetranitromethane

Yumiko OHTA*, Sachiko TANABE**, Akira HACHIMORI and Tatsuya SAMEJIMA

Department of Chemistry, College of Science and Engineering, Aoyama Gakuin University 6-16-1 Chitosedai, Setagaya-ku, Tokyo 157

Thermolysin [EC 3.4.24.4 [EC] ] was nitrated with tetranitromethane (TNM) to elucidate the contribution of tyrosyl residues to its thermostability and enzyme action. The nitration of 3 and 6 tyrosyl residues caused a reduction of the enzyme activity to 50% and 35%, respectively. Kinetic analysis indicated that the Km value of the nitrated enzyme for benzyloxycarbonyl-Gly-Leu-NH2 remained unchanged, whereas the maximum velocity decreased on nitration compared with that of the native enzyme. Heat treatment at 80°C for 30 min revealed that nitrated thermolysin is less thermostable than the native enzyme. The {alpha}-helical conformation was found to be unaffected by nitration on the basis of ORD analysis. The nitrated thermolysin did not show fluorescence due to tryptophanyl residues, indicating that tryptophanyl residues had reacted with TNM, which caused quenching of their fluorescence. This view was supported by the spectrophotometric determination of tryptophan. It was inferred that the inactivation of the enzyme is due to the nitration of tyrosyl residues rather than tryptophanyl residues.

*Present address: Nippon Roche Research Center, Kajiwara, Kamakura, Kanagawa.

**Present address: Department of Physics, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo.


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