We have performed site directed mutagenesis a the enzyme adenylate kinase (AK) from E. Coli. The four tryptophan mutants (W 41, 86, 133, 137) were over expressed in good yield. All the mutants were active, but with reduced levels of activity vis-a-vis the wild type.
The specific aim of the research is to examine how the point mutants change the dynamics of AK folding and ligand binding without gross perturbation of the enzyme's activity or native conformation. Steady-state fluorescence indicates large wavelengths shifts in emission (~10-15 nm) in going from W41 to W137. Presumably, these shifts relate to the location of the tryptophan residue: buried or exposed. Lifetime measurements support this interpretation, as the values range from 2 to 8 nsec. Fluorescence quenching experiments provide further corroboration for this model. Future experiments will deal with the kinetics of AK folding and ligand binding.
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