Structure‐Activity Relationship in Tryptophanyl‐Transfer Ribonucleic Acid Synthetase from Beef Pancreas

The titration of the – SH groups of beef pancreas tryptophanyl‐tRNA synthetase by N‐ ethylmaleimide and the consequent effect on the exchange and transfer reactions of the enzyme were examined. The 16 sulfhydryl groups of the native dimeric enzyme do not react homogeneously at pH 7, with 1 mM reagent. They can be divided into three classes with decreasing reactivities. The reaction of the first class (6 – SH groups) has no effect on activity. The reaction of the second class (4 – SH groups) brings about an increase of K m(app) for tryptophan both in the exchange and in the transfer reactions of the enzyme. The activity of the enzyme is lost during reaction of the third class (6 ‐ SH groups). This is evidenced by the decrease of the V of the [ 32 P]PP i ‐ATP isotope exchange and tRNA aminoacylation reactions and by the decrease in the amount of the tryptophanyladenylate · enzyme complex isolatable on Biogel P30. The destruction of the active site by alkylation of the sulfhydryl groups of class III can be the consequence of a structural change of the enzyme induced by the chemical modification. During the alkylation of the first two classes of ‐ SH groups the K m(app) for ATP, PP i and tRNA are not altered significantly. The presence of tryptophan or tryptophanyladenylate reduces the rate of titration of the ‐SH groups of class II and class III and in a parallel fashion the rates of increase of K m(app) for tryptophan and of decrease of V. The titration of the –SH groups of class I is not affected by the presence of substrates. The alkylation in the presence of tryptophanyladenylate demonstrates that class II is not homogeneous and that the two molecules of adenylate bound to the dimeric enzyme specifically protect two –SH groups out of the four of this class.