EFFECTS OF NITRATION AND AMINATION OF TYROSYL RESIDUES IN THERMOLYSINON ITS HYDROLYTIC ACTIVITY AND ITS REMARKABLE ACTIVATION BY SALTS

Thermolysin is remarkably activated in the presence of high concentrat ions (1-5 M) of neutral salts and its activity is enhanced 15 times by 4 M NaCl at pH 7.0 and 25 degrees C [Inouye, K. (1992) J. Biochem, 11 2, 335-340], In this study, the effects of nitration and amination of tyrosyl residues in thermolysin on its halophilic properties were exam ined. Nitration and successive amination inactivate thermolysin progre ssively as the degree of modification increases. When 16 tyrosyl resid ues were nitrated, the activity decreased to 10% of that of the native enzyme, whereas it recovered to 30% when they mere aminated, The decr ease in the activity by the nitration and amination was shown to be br ought about only by a decrease in the molecular activity, k(cat); the Michaelis constant, K-m, was unaltered. When 14 tyrosyl residues of th ermolysin were nitrated, the degree of activation by ii Ri 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 nitrate d tyrosyl residues were reduced to aminotyrosyl residues, the degree o f activation was restored to that of the native enzyme, The change in the degree of activation by nitration and amination of thermolysin cou ld 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.