Navegando por Palavras-chave "binding energy"

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    Subsites of trypsin active site favor catalysis or substrate binding
    (Elsevier B.V., 2002-01-11) Marana, SR; Lopes, A. R.; Juliano, L.; Juliano, M. A.; Ferreira, C.; Terra, W. R.; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)
    Enzymes enhance chemical reaction rates by lowering the activation energy, the energy barrier of the reaction leading to products. This occurs because enzymes bind the high-energy intermediate of the reaction (the transition state) more strongly than the substrate. We studied details of this process by determining the substrate binding energy (DeltaG(g), calculated from K-m values) and the activation energy (DeltaG(T), determined from k(cat)/K-m values) for the trypsin-catalyzed hydrolysis of oligopeptides. Plots of DeltaG(T) versus DeltaG(g) for oligopeptides with 15 amino acid replacements at each of the positions P-1', P-1, and P-2 were straight lines, as predicted by a derived equation that relates DeltaG(T) and DeltaG(g). the data led to the conclusion that the trypsin active site has subsites that bind moieties of substrate and of transition state in characteristic ratios, whichever substrate is used. This was unexpected and means that each subsite characteristically favors substrate binding or catalysis. (C) 2002 Elsevier Science.
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    Substrate specificity of insect trypsins and the role of their subsites in catalysis
    (Elsevier B.V., 2006-02-01) Lopes, A. R.; Juliano, M. A.; Marana, SR; Juliano, L.; Terra, W. R.; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)
    Trypsins have high sequence similarity, although the responses of insect trypsins to chemical and natural inhibitors suggest they differ in specificities. Purified digestive trypsins from insects of four different orders were assayed with internally quenched fluorescent oligopeptides with two different amino acids at P1 (Arg/Lys) and 15 amino acid replacements in positions P1', P2, P2, and P3. the binding energy (Delta G(s), calculated from K-m values) and the activation energy (Delta G(T)(++), determined from k(cat)/K-m values) were calculated. Dictyoptera, Coleoptera and Diptera trypsins hydrolyze peptides with Arg at P, at least 3 times more efficiently than peptides with Lys at P1, whereas Lepidoptera trypsins have no preference between Arg and Lys at that position. the hydrophobicities of each subsite were calculated from the efficiency of hydrolysis of the different amino acid replacements at that subsite. the results suggested that insect trypsin subsites become progressively more hydrophobic along evolution. Apparently, this is an adaptation to resist plant protein inhibitors, which usually have polar residues at their reactive sites. Results also suggested that, at least in lepidopteran trypsins, S3, S2, S1' and S2' significantly bind the substrate ground state, whereas in the transition state only S1' and S2' do that, supporting aspects of the presently accepted mechanism of trypsin catalysis. Homology modeling showed differences among those trypsins that may account for the varied kinetic properties (c) 2005 Elsevier B.V. All rights reserved.