Thermodynamic analysis reveals structural rearrangement during the acylation step in human trypsin 4 on 4-methylumbelliferyl 4-guanidinobenzoate substrate analogue

Júlia Tóth, Linda Gombos, Zoltán Simon, Péter Medveczky, László Szilágyi, L. Gráf, A. Málnási-Csizmadia

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Abstract

Human trypsin 4 is an unconventional serine protease that possesses an arginine at position 193 in place of the highly conserved glycine. Although this single amino acid substitution does not affect steady-state activity on small synthetic substrates, it has dramatic effects on zymogen activation, interaction with canonical inhibitors, and substrate specificity toward macromolecular substrates. To study the effect of a non-glycine residue at position 193 on the mechanism of the individual enzymatic reaction steps, we expressed wild type human trypsin 4 and its R193G mutant. 4-Methylumbelliferyl 4-guanidinobenzoate has been chosen as a substrate analogue, where deacylation is rate-limiting, and transient kinetic methods were used to monitor the reactions. This experimental system allows for the separation of the individual reaction steps during substrate hydrolysis and the determination of their rate constants dependably. We suggest a refined model for the reaction mechanism, in which acylation is preceded by the reversible formation of the first tetrahedral intermediate. Furthermore, the thermodynamics of these steps were also investigated. The formation of the first tetrahedral intermediate is highly exothermic and accompanied by a large entropy decrease for the wild type enzyme, whereas the signs of the enthalpy and entropy changes are opposite and smaller for the R193G mutant. This difference in the energetic profiles indicates much more extended structural and/or dynamic rearrangements in the equilibrium step of the first tetrahedral intermediate formation in wild type human trypsin 4 than in the R193G mutant enzyme, which may contribute to the biological function of this protease.

Original languageEnglish
Pages (from-to)12596-12602
Number of pages7
JournalJournal of Biological Chemistry
Volume281
Issue number18
DOIs
Publication statusPublished - May 5 2006

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Acylation
Thermodynamics
Structural analysis
Trypsin
Entropy
Substrates
Enzyme Precursors
Serine Proteases
Enzymes
Amino Acid Substitution
Substrate Specificity
Glycine
Arginine
Hydrolysis
Peptide Hydrolases
Enthalpy
Rate constants
Substitution reactions
Chemical activation
4-guanidinobenzoate

ASJC Scopus subject areas

  • Biochemistry

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Thermodynamic analysis reveals structural rearrangement during the acylation step in human trypsin 4 on 4-methylumbelliferyl 4-guanidinobenzoate substrate analogue. / Tóth, Júlia; Gombos, Linda; Simon, Zoltán; Medveczky, Péter; Szilágyi, László; Gráf, L.; Málnási-Csizmadia, A.

In: Journal of Biological Chemistry, Vol. 281, No. 18, 05.05.2006, p. 12596-12602.

Research output: Contribution to journalArticle

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