STUDY OF
ENZYME CATALYSIS USING TRANSIENT KINETIC AND MICROCALORIMETRY TECHNIQUES
Z. Prokop1, M. Monincová1,
R. Chaloupková1, Y. Nagata2, D.B. Janssen3,
J. Damborský1
1National Centre for Biomolecular Research,
Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic, 2Department of Life Sciences, Graduate School of
Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai, 980-8577, Japan, 3University
of Groningen, Nijenborgh 4, 9747 AG Groningen, The Nedherlands
Kinetic studies in enzymology deals
with elucidation of enzymatic reaction pathway, identification of reaction
intermediates and with specification of the steps that limit the rate of
turnover. The kinetic analysis of an enzyme mechanism often begins by analysis
of the steady-state kinetics. The steady-state kinetic parameters, Km
and kcat, provide information sufficient to describe a
minimal kinetic scheme. Conclusions that can be derived by steady-state
analysis are considered preliminary. Because the steady-state kinetic
parameters are complex functions of all the reactions occurring during enzymatic
catalysis, individual reaction steps are buried within these terms and cannot
be resolved. These limitations are overcome by examination of the reaction
pathway by transient kinetic methods, where the enzyme is examined as a
stoichiometric reactant, allowing individual steps in pathway to be established
by direct measurement. Steady-state and transient-state kinetic studies
complement each other in elucidation of enzymatic reaction pathway. Analysis in
the steady-state should be a prelude to the proper design and interpretation
for more detailed transient-state experiments [1].
Both steady-state and
transient-state kinetic methods were applied to solve reaction pathway, to
identify reaction intermediate and to specify the rate limiting step of catalytic
action of haloalkane dehalogenase LinB from bacterial strain Sphingomonas
paucimobilis UT26 [2]. Steady-state experiments involved direct monitoring
of LinB activity by isothermal titration calorimetry and initial rate of
product formation measurements by gas chromatography. Transient-state kinetics
used stopped-flow fluorescence and rapid-quench-flow techniques. Additional
steady-state inhibition experiments and transient-state binding experiments
were employed to find out leaving ability of both products (a halide and a
alcohol) during dehalogenation reaction.
The results showed that export of
products as well as import of substrates into the active site of LinB are fast
processes reaching rapid equilibrium. This fast exchange of the ligands between
the active site and bulk solvent can be explained by wide opening of the
entrance tunnel and large active site of LinB. In contrary, the release of the
halide ion from narrow active site after the reaction was found to be slow rate
limiting step for another haloalkane dehalogenase, enzyme DhlA from Xanthobacter
autotrophicus GJ10 [3]. The actual cleavage of the carbon-halogen bond was
found to be fast step in both enzymes.
The rate of cleavage of C-Br bound is faster than cleavage of C-Cl bound, which is in agreement with bromide being a better leaving group in biomolecular nucleophilic substitution than chloride. This observation correlates with the lower affinity of LinB for chloride compared to bromide. Further the results confirmed, that the reaction proceeds via a covalent alkyl-enzyme intermediate. Using bromocyclohexane, chlorocyclohexane and 1-chlorohexane as model substrates, hydrolysis of this intermediate was found to be the slowest step in the catalytic cycle of LinB. The alkyl-enzyme complex was highly accumulated due to the fast dehalogenation step following the slow hydrolyses of this intermediate. The study provides a basis for the analysis of kinetic steps in hydrolysis of environmentally important substrates by the action of LinB.
[1] K.A. Johnson : Enzymes 20. Academic Press 1992. 1-61.
[2] Y. Nagata, K. Miyauchi, J. Damborsky, K. Manova, A., Ansorgova & M. Takagi, Appl. Environ. Microb., 63 (1997) 3707-3710.
[3] J. P. Schanstra, J. Kingma & D. B. Janssen, J. Biol. Chem., 271 (1996) 14747-14753.