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A fluorescence stopped-flow kinetic study of the conformational activation of alpha-chymotrypsin and several mutants.

ABSTRACT: The kinetic activation parameters (activation free energy, activation free enthalpy, and activation free entropy change) of the conformational change of alpha-chymotrypsin from an inactive to the active conformation were determined after a pH jump from pH 11.0 to pH 6.8 by the fluorescence stopped-flow method. The conformational change was followed by measuring changes in the protein fluorescence. For the bovine wild-type protein, the same kinetic parameters are obtained as in the study of proflavin binding. Several mutants were made with the goal to accelerate or decelerate this conformational transition. The inspiration for the choice of the mutants came from a previous modelling study done on the bovine wild-type chymotrypsin. The results of the fluorescence stopped flow experiments show that several mutants behaved as was expected based on the information provided by the modeling study on the wild-type variant. For some mutants our assumptions were not correct, and therefore additional modeling studies of the activation pathways of these mutant proteins are necessary to be able to explain the observed kinetic behavior.

SUBMITTER: Verheyden G

PROVIDER: S-EPMC2280002 | biostudies-literature | 2004 Sep

REPOSITORIES: biostudies-literature

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A fluorescence stopped-flow kinetic study of the conformational activation of alpha-chymotrypsin and several mutants.

Verheyden Gert G Matrai Janka J Volckaert Guido G Engelborghs Yves Y

Protein science : a publication of the Protein Society 20040901 9

The kinetic activation parameters (activation free energy, activation free enthalpy, and activation free entropy change) of the conformational change of alpha-chymotrypsin from an inactive to the active conformation were determined after a pH jump from pH 11.0 to pH 6.8 by the fluorescence stopped-flow method. The conformational change was followed by measuring changes in the protein fluorescence. For the bovine wild-type protein, the same kinetic parameters are obtained as in the study of profl ...[more]

PMID: 15322291

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Project description:Lon is an ATP dependent serine protease responsible for degrading denatured, oxidatively damaged and certain regulatory proteins in the cell. In this study we exploited the fluorescence properties of a dansylated peptide substrate (S4) and the intrinsic Trp residues in Lon to monitor peptide interacting with the enzyme. We generated two proteolytically inactive Lon mutants, S679A and S679W, where the active site serine is mutated to an Ala and Trp residue, respectively. Stopped-flow fluorescence spectroscopy was used to identify key enzyme intermediates generated along the reaction pathway prior to peptide hydrolysis. A two-step peptide binding event is detected in both mutants, where a conformational change occurs after a rapid equilibrium peptide binding step. The Kd for the initial peptide binding step determined by kinetic and equilibrium binding techniques is approximately 164 micromolar and 38 micromolar, respectively. The rate constants for the conformational change detected in the S679A and S679W Lon mutants are 0.74 +/- 0.10 s(-1) and 0.57 +/- 0.10 s(-1), respectively. These values are comparable to the lag rate constant determined for peptide hydrolysis (klag approximately 1 s(-1)) [Vineyard, D., et al. (2005) Biochemistry 45, 4602-4610]. Replacement of the active site Ser with Trp (S679W) allows for the detection of an ATP-dependent conformational change within the proteolytic site. The rate constant for this conformational change is 7.6 +/- 1.0 s(-1), and is essentially identical to the burst rate constant determined for ATP hydrolysis under comparable reaction conditions. Collectively, these kinetic data support a mechanism by which the binding of ATP to an allosteric site on Lon activates the proteolytic site. In this model, the energy derived from the binding of ATP minimally supports peptide cleavage by allowing peptide substrate access to the proteolytic site. However, the kinetics of peptide cleavage are enhanced by the hydrolysis of ATP.

Dataset Information

A fluorescence stopped-flow kinetic study of the conformational activation of alpha-chymotrypsin and several mutants.

Publications

A fluorescence stopped-flow kinetic study of the conformational activation of alpha-chymotrypsin and several mutants.

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