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 reaction with p-diazoniumbenzamidine chloride was confirmed by spectral analysis. Proteolysis of Cm-p-benzamidino-azo-beta-trypsin provided peptides containing modified tyrosine residues. The major product, Ser-146 to Lys-156, which corresponded to labeling at Tyr-151, was recovered in 35% yield, and its structure was demonstrated by amino acid analysis, Edman degradation, and mass spectrometry. Yields of labeled Tyr-151, Tyr-39, and Tyr-172, identified by peptide analysis, were in the proportion of 100:7:3. Tyr-151-(p-benzamidino)-azo-beta-trypsin is permanently activated, but can be further activated by substrates. Values of kcat, Ks', and kcat' vary from two to three times the equivalent values for trypsin. Berenil (4,4'-diazoamino-bis-benzamidine), a parabolic competitive inhibitor of beta-trypsin, was a hyperbolic competitive inhibitor of azo-beta-trypsin. Thus, Tyr-151, part of subsite S'2, affects the catalytic process and, when modified covalently, permanently activates trypsin. Equilibrium binding with berenil supported the kinetic data obtained with substrates. This permits the integration of protein modification, kinetics, equilibrium binding, and crystallographic data to demonstrate a fine interaction between subsites S1-S3 and S'2 in trypsin and azo-beta-trypsin, resulting in subtle structural changes when the native enzyme is covalently modified at Tyr-151.
Oliveira
M G
MG
Department of Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, Brazil.
Rogana
E
E
Rosa
J C
JC
Reinhold
B B
BB
Andrade
M H
MH
Greene
L J
LJ
Mares-Guia
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001550:Benzamidines; D001665:Binding Sites; D002417:Cattle; D003979:Diazonium Compounds; D004789:Enzyme Activation; D007700:Kinetics; D008969:Molecular Sequence Data; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*
D001550:Benzamidines; D003979:Diazonium Compounds; D014361:Trypsin Inhibitors; C084966:Tyr(151)-4-benzamidinoazo-beta-trypsin; D014443:Tyrosine; C084986:4-diazoniumbenzamidine; D014357:Trypsin