A fluorescence stopped‐flow kinetic study of the conformational activation of α‐chymotrypsin and several mutants

Verheyden, Gert; Mátrai, Janka; Volckaert, Guido; Engelborghs, Yves

doi:10.1110/ps.04709604

Cited by 10 publications

(15 citation statements)

References 27 publications

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“…1). The reaction can be followed by the change of the intrinsic tryptophan fluorescence in a stoppedflow apparatus (11,15). At high pH, the active trypsin acquires the zymogen conformation, which is transformed to the active form on pH reduction.…”

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confidence: 99%

“…At high pH, the active trypsin acquires the zymogen conformation, which is transformed to the active form on pH reduction. The reaction can be followed by the change of the intrinsic tryptophan fluorescence in a stoppedflow apparatus (11,15). The zymogen and the active forms are stable conformations of trypsinogen and trypsin, respectively, and their atomic crystal structures are available (1TGN, 2PTN).…”

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confidence: 99%

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Temperature dependence of internal friction in enzyme reactions

Rauscher

Simon

Szöllősi³

et al. 2011

FASEB j.

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Our aim was to elucidate the physical background of internal friction of enzyme reactions by investigating the temperature dependence of internal viscosity. By rapid transient kinetic methods, we directly measured the rate constant of trypsin 4 activation, which is an interdomain conformational rearrangement, as a function of temperature and solvent viscosity. We found that the apparent internal viscosity shows an Arrhenius-like temperature dependence, which can be characterized by the activation energy of internal friction. Glycine and alanine mutations were introduced at a single position of the hinge of the interdomain region to evaluate how the flexibility of the hinge affects internal friction. We found that the apparent activation energies of the conformational change and the internal friction are interconvertible parameters depending on the protein flexibility. The more flexible a protein was, the greater proportion of the total activation energy of the reaction was observed as the apparent activation energy of internal friction. Based on the coupling of the internal and external movements of the protein during its conformational change, we constructed a model that quantitatively relates activation energy, internal friction, and protein flexibility.

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confidence: 99%

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confidence: 99%

Temperature dependence of internal friction in enzyme reactions

Rauscher

Simon

Szöllősi³

et al. 2011

FASEB j.

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“…The conformational change upon activation can also be triggered by a pH‐jump from pH 11.0 to pH 8.07–9 and monitored by measuring the intrinsic fluorescence of the enzyme,10 offering an elegant experimental approach to study the structural rearrangement. It was shown for chymotrypsin that the rate constants measured by the pH‐jump method are in excellent agreement with the data acquired from proflavine binding studies 10, 11…”

Section: Introductionmentioning

confidence: 99%

“…These glycine residues exhibit larger Φ and/or Ψ angle changes than the surrounding residues. This suggests that these glycines have a well‐defined role in the activation process: they act as hinges for the conformational change and the four peptide segments move as more rigid units 10, 12. The presence of glycines at conserved positions of the activation domain seems to promote the conformational transition.…”

Section: Introductionmentioning

confidence: 99%

Site directed mutagenesis at position 193 of human trypsin 4 alters the rate of conformational change during activation: Role of local internal viscosity in protein dynamics

et al. 2007

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Upon activation of trypsinogen four peptide segments flanked by hinge glycine residues undergo conformational changes. To test whether the degree of conformational freedom of hinge regions affects the rate of activation, we introduced amino acid side chains of different characters at one of the hinges (position 193) and studied their effects on the rate constant of the conformational change. This structural rearrangement leading to activation was triggered by a pH-jump and monitored by intrinsic fluorescence change in the stopped-flow apparatus. We found that an increase in the size of the side chain at position 193 is associated with the decrease of the reaction rate constant. To analyze the thermodynamics of the reaction, temperature dependence of the reaction rate constants was examined in a wide temperature range (5-60 degrees C) using a novel temperature-jump/stopped-flow apparatus developed in our laboratory. Our data show that the mutations do not affect the activation energy (the exponential term) of the reaction, but they significantly alter the preexponential term of the Arrhenius equation. The effect of solvent viscosity on the rate constants of the conformational change during activation of the wild type enzyme and its R193G and R193A mutants was determined and evaluated on the basis of Kramers' theory. Based on this we propose that the reaction rate of this conformational transition is regulated by the internal molecular friction, which can be specifically modulated by mutagenesis in the hinge region.

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“…It allows the formation of a salt bridge between the newly released Ile 16 N terminus and the Asp 194 carboxyl group. This activation of the zymogen can also be mimicked by a pH jump from an inactive chymotrypsinogen‐like structure at high pH to an active conformation around pH 7 and so can be followed spectroscopically (Fersht and Renard 1974; Stoesz and Lumry 1978; Heremans and Heremans 1989a,b; Verheyden et al 2004).…”

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Simulation of the activation of α‐chymotrypsin: Analysis of the pathway and role of the propeptide

et al. 2004

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Abstract␣-Chymotrypsin undergoes a reversible conformational change from an inactive chymotrypsinogen-like structure at high pH to an active conformation at neutral pH. In order to gain insight into this process on a structural level, we applied molecular dynamics and targeted molecular dynamics simulations in aqueous environment on the activation and inactivation processes of three different types of chymotrypsin. These are the wild-type bovine chymotrypsin containing the propeptide and the bovine and rat chymotrypsin lacking the propeptide. From these simulations, the importance of the propeptide and of the sequence differences between the rat and bovine variants from the viewpoint of activation could be evaluated and compared with previous fluorescence stopped flow results. The obtained results show the unambiguous influence of the propeptide on the explored conformational space, whereas the sequence differences between bovine and rat chymotrypsin play a minor role. The main features of activation are present in both the wild type and the variant lacking the propeptide, despite the fact that different parts of the conformational space were explored. The comparison of all trajectories shows that particular amino acid residues, such as 17, 18, 19, 187, 217, 218, and 223, undergo large dihedral transitions during the activation process, suggesting a role as hinge residues during the conformational change.Keywords: conformational change; molecular dynamics simulation; targeted molecular dynamics; pathway calculation; chymotrypsinogen; chymotrypsin; A-chain (propeptide); fluorescence stopped flow Supplemental material: see www.proteinscience.org Conformational changes play an essential role in the modulation of biological activity in proteins. Proteases are synthesized as inactive proenzymes, zymogens, and perform a series of conformational changes toward their active conformation in order to provide a site-and time-specific regulation of their proteolytic activity. The inactive protein precursors often consist of the intact protease with an N-terminal extension or prosegment, stabilizing the inactive enzyme conformation. Activation of the enzyme is then accomplished by limited cleavage of the propeptide in the zymogen. A typical example is the activation of chymotrypsinogen to chymotrypsin occurring after tryptic cleavage of the peptide bond between amino acid residues 15 and 16. This cleavage triggers a series of conformational changes that activates the enzyme (Bode et al. 1978;Bode 1979;Wang et al. 1985;Wroblowski et al. 1997). It allows the formation of a salt bridge between the newly released Ile 16 N terminus and the Asp 194 carboxyl group. This activation of the zymogen can also be mimicked by a pH jump from an inactive chymotrypsinogen-like structure at high pH to an active conformation around pH 7 and so can be followed spectroscopically (Fersht and Renard 1974 Abbreviations: rms, root mean square; MD, molecular dynamics; rmsd, root mean square deviation; TMD, targeted molecular dynamics; ⌬A-chymotrypsi...

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A fluorescence stopped‐flow kinetic study of the conformational activation of α‐chymotrypsin and several mutants

Cited by 10 publications

References 27 publications

Temperature dependence of internal friction in enzyme reactions

Temperature dependence of internal friction in enzyme reactions

Site directed mutagenesis at position 193 of human trypsin 4 alters the rate of conformational change during activation: Role of local internal viscosity in protein dynamics

Simulation of the activation of α‐chymotrypsin: Analysis of the pathway and role of the propeptide

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