TYROSINE-151 IS PART OF THE SUBSTRATE ACTIVATION BINDING-SITE OF BOVINE TRYPSIN - IDENTIFICATION BY COVALENT LABELING WITH P-DIAZONIUMBENZAMIDINE AND KINETIC CHARACTERIZATION OF TYR-151-(P-BENZAMIDINO)-AZO-BETA-TRYPSIN

Identification of the substrate activation site of beta-trypsin by a 1 :1 reacton with p-diazoniumbenzamidine chloride was confirmed by spect ral anaylsis. Proteolysis of Cm-p-benzamidino-azo-beta-trypsin provide d peptides containing modified tyrosine residues. The major product, S er-146 to Lys-156, which corresponded to labeling at Tyr-151, was reco vered in 35% yield, and its structure was demonstrated by amino acid a nalysis, Edman degradation, and mass spectrometry. Yields of labeled T yr-151, Tyr-39, and Tyr-172, identified by peptide analysis, were in t he proportion of 100:7:3. Tyr-151-(p-benzamidino)-azo-beta-trypsin is permanently activated, but can be further activated by substrates. Val ues of k(cat), Ks', and k(cat) vary from two to three times the equiva lent values for trypsin. Berenil (4,4'-diazoamino-bis-benzamidine), a parabolic competitive inhibitor of beta-trypsin, was a hyperbolic comp etitive inhibitor of azo-beta-trypsin. Thus, Tyr-151, part of subsite S'2, affects the catalytic process and, when modified covalently, perm anently activates trypsin. Equilibrium binding with berenil supported the kinetic data obtained with substrates. This permits the integratio n of protein modification, kinetics, equilibrium binding, and crystall ographic data to demonstrate a fine interaction between subsites S1-S3 and S'2 in trypsin and azo-beta-trypsin, resulting in subtle structur al changes when the native enzyme is covalently modified at Tyr-151.