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|Title:||Tyrosine 151 is part of the substrate activation binding site of bovine trypsin.|
|Authors:||Oliveira, Maria Goreti de Almeida|
Rosa, José César
Reinhold, Bruce B.
Andrade, Milton Hércules Guerra de
Greene, Lewis Joel
Guia, Marcos Luíz dos Mares
|Citation:||OLIVEIRA, M. G. de A. et al. Tyrosine 151 is part of the substrate activation binding site of bovine trypsin. The Journal of Biological Chemistry, v. 268, p. 26893-26903, 1993. Disponível em: <http://www.jbc.org/content/268/36/26893.long>. Acesso em: 23 fev. 2017.|
|Abstract:||Identification of the substrate activation site of p-trypsin by a 1:l reacwtiotnh p-diazoniumbenzamidine chloride was confirmebdy spectral anaylsisP. roteolysis of Cm-p-benzamidino-azo-@-trypsipnr ovided peptides containing modified tyrosine residues. The major product, Ser-146 to Lys-156, which corresponded to labeling at '&r-151, was recovereidn 35%y ield, and its structure was demonstrated by amino acid analysis, Edman deg radation, and mass spectrometry. Yielodfs labeled Tyr- 151, Tyr-39, and Tyr-172,i dentified by peptide analysis, were in the proportion of 100:7:3. Tyr-l51-(p-benzamidinol- azo-j3-trypsinis permanently activated, but can be further activated by substrates. Values of kcat, Ks', and kc,,. vary from twot o three times the equivalent values for trypsin. Berenil (4,4'-diazoamino-bis-benzamidine), a parabolic competitive inhibitoorf ptrypsin, was ah yperbolic competitive inhibitor of azo-p-trypsin. Thus, Tyr-151, part of subsite 5'2, affects the catalytic process and, when modified covalently, permanently activates trypsin. Equilibrium bindinwgi th berenil supportedt he kinetic data obtainedw ith substrates. This permitst he integration of protein modification, kinetics, equilibrium binding, and crystallographic datao demonstrate a fine interaction between subsites S1-S3 and5 '2 in trypsin and azo-p-trypsin, resulting in subtle structural changes when the native enzyme is covalently modified at Qr-151.|
|Appears in Collections:||DECBI - Artigos publicados em periódicos|
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