During my thesis work I had a chance to join a developer team planning a microcalorimeter device. My aim was to express, purify and characterize phenylalanine ammonia lyase (PAL) and deoxyuridine-triphosphatase (dUTPase) enzymes, as they are promising candidates for diagnostic purposes. Both active and inactive forms of the enzymes were expressed with E. coli strains. The inactive mutants were planned to be not perturbed in substrate-binding but inhibited in conversion, being suitable for immobilization, isothermal titration calorimeter, thermostability and activity measurements. I considered not only the diagnostic potential, but the kinetic data and the structural stability properties based on the protein structures to choose the most promising candidates. After cell disruption the proteins were purified with Ni-NTA affinity and enzyme activity measurements were performed. I investigated enzyme activity dependence from the substrate concentration of the PAL enzymes to determine the kinetic constants. Further investigations included:
• Enzyme activity - pH dependence
• Thermostability of the enzymes
PAL structures based on homology modeling and the PAL catalytic mechanism were investigated and in substrate-binding not perturbed but in conversion inhibited modified enzymes were planned. These mutants were expressed and investigated the same way (methods) as the native enzymes. These inactive mutants could serve as references in blind measurements, as the friction heat from substrate binding is approximately the same as by the native enzymes and the catalysis can not be performed. I also prepared high amounts of purified proteins for immobilization purposes.
A special chimera PAL enzyme was also expressed and investigated. This chimera was planned based on the available bioinformatics data. We replaced the eukaryotic C-terminal multihelix region with a prokaryotic segment in a promising eukaryotic PcPAL candidate.