Effects of nitration and amination of tyrosyl residues in thermolysin on its hydrolytic activity and its remarkable activation by salts

Kuniyo Inouye, Soo-Bok Lee, Ben'ichiro Tonomura

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Thermolysin is remarkably activated in the presence of high concentrations (1-5 M) of neutral salts and its activity is enhanced 15 times by 4 M NaCl at pH 7.0 and 25°C. In this study, the effects of nitration and amination of tyrosyl residues in thermolysin on its halophilic properties were examined. Nitration and successive amination inactivate thermolysin progressively as the degree of modification increases. When 16 tyrosyl residues were nitrated, the activity decreased to 10% of that of the native enzyme, whereas it recovered to 30% when they were aminated. The decrease in the activity by the nitration and amination was shown to be brought about only by a decrease in the molecular activity, k(cat); the Michaelis constant, K(m), was unaltered. When 14 tyrosyl residues of thermolysin were nitrated, the degree of activation by 4 M NaCl at pH 7.0 decreased from 15 to 10, and this decreased further to 5 when the pH of the reaction medium was raised to 8.5. However, when the nitrated tyrosyl residues were reduced to aminotyrosyl residues, the degree of activation was restored to that of the native enzyme. The change in the degree of activation by nitration and amination of thermolysin could be due to the change in the ionization of tyrosyl residues, and it was suggested that removing negative charges from tyrosyl residues of thermolysin enhances its halophilicity.

Original languageEnglish
Pages (from-to)72-78
Number of pages7
JournalJournal of Biochemistry
Volume124
Issue number1
DOIs
Publication statusPublished - 1998 Jan 1

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Thermolysin
Nitration
Amination
Salts
Chemical activation
Enzymes
Ionization
Thermodynamic properties

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

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title = "Effects of nitration and amination of tyrosyl residues in thermolysin on its hydrolytic activity and its remarkable activation by salts",
abstract = "Thermolysin is remarkably activated in the presence of high concentrations (1-5 M) of neutral salts and its activity is enhanced 15 times by 4 M NaCl at pH 7.0 and 25°C. In this study, the effects of nitration and amination of tyrosyl residues in thermolysin on its halophilic properties were examined. Nitration and successive amination inactivate thermolysin progressively as the degree of modification increases. When 16 tyrosyl residues were nitrated, the activity decreased to 10{\%} of that of the native enzyme, whereas it recovered to 30{\%} when they were aminated. The decrease in the activity by the nitration and amination was shown to be brought about only by a decrease in the molecular activity, k(cat); the Michaelis constant, K(m), was unaltered. When 14 tyrosyl residues of thermolysin were nitrated, the degree of activation by 4 M NaCl at pH 7.0 decreased from 15 to 10, and this decreased further to 5 when the pH of the reaction medium was raised to 8.5. However, when the nitrated tyrosyl residues were reduced to aminotyrosyl residues, the degree of activation was restored to that of the native enzyme. The change in the degree of activation by nitration and amination of thermolysin could be due to the change in the ionization of tyrosyl residues, and it was suggested that removing negative charges from tyrosyl residues of thermolysin enhances its halophilicity.",
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Effects of nitration and amination of tyrosyl residues in thermolysin on its hydrolytic activity and its remarkable activation by salts. / Inouye, Kuniyo; Lee, Soo-Bok; Tonomura, Ben'ichiro.

In: Journal of Biochemistry, Vol. 124, No. 1, 01.01.1998, p. 72-78.

Research output: Contribution to journalArticle

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