| Relative dehalogenating activities were measured with different haloalkane dehalogenases for more then 160 halogenated substrates (Damborsky, et al., 2001). These data however are not homogenous because they originates from different laboratories and by definition they provide only limited information about actual activity and affinity of dehalogenases towards these substrates. Steady-state and pre-steady state kinetic data are much more suitable for characterisation of catalytic properties of enzymes, but these are available only for a limited number of substrates and selected dehalogenases. Pre-steady state kinetics will be studied for haloalkane dehalogenase LinB. Kinetic rates of separate catalytic steps will be directly comparable with the data obtained from the quantum-mechanic calculations and can be used for the validation purposes. Identification of the rate-limiting step for a particular substrate is also needed for the design of enzymes with enhanced activities. Microcalorimetric measurements will be used for determination of thermodynamic parameters for binding of substrates to the protein active site and will serve as the input data for Comparative Binding Energy analysis. CD-spectroscopy will be applied for a fast structural and stability characterisation of constructed point mutants and hybrid enzymes. Crystallographic analysis will be conducted for study of the proteins with most interesting catalytic properties and for the analysis of protein-ligand complexes. |
