REACTION OF TRYPSIN WITH BROMOACETONE

The reaction of trypsin and certain trypsin derivatives with bromoacetone has been investigated. The resultant inactivation of the enzyme, measured by the rate of hydrolysis of ester substrates or by reaction with [32P]diisopropylphosphofluoridate ([32P]DFP), correlated with loss of one histidine residue. Histidine also reacted in diisopropylphosphoryl-(DIP) trypsin but not in L-1-chloro-2-tosylamido-7-amino-2-heptanone (TLCK) trypsin. Experiments were also carried out using [1,3-14C]bromoacetone. A chromatographic procedure, based on the high affinity of trypsin for soybean trypsin inhibitor, was employed to separate active from inactive trypsin in the reaction mixture. The inactivated trypsin contained 1.06 moles of [14C]-acetone more than did the active material and showed a loss of 0.8 residue of histidine when compared with the internal control. Thus inactivation resulted from modification of a single residue of histidine. The radioactive peptides present in the inactive but not in the active bromoactone-treated trypsin contained 1.1 moles of I4C and included histidine-46, while the peptide including histidine-27 contained a negligible quantity of 14C. Bromoacetone was also found to react almost exclusively with histidine-46 in DIP-trypsin. Thus of the three histidine residues present in trypsin, only histidine-46 reacted to a significant extent with bromoacetone. The rate of reaction of histidine-46 in trypsin was comparable to that of the model compound α-N-ben-zoyl-L-histidine methyl ester. Furthermore, the introduction of an acetonyl group into histidine-46 abolished the unusual reactivity of serine-183, thus providing chemical evidence in support of the hypothesis that interaction between these residues is essential for the catalytic function of trypsin. © 1968, American Chemical Society. All rights reserved.