Phenylketonuria (PKU; OMIM 261600), the most common disorder of
amino acid metabolism, is caused by a deficient activity of human
phenylalanine hydroxylase (
hPAH). Although the dietetic treatment has proven to be effective in preventing the psycho-motor impairment, much effort has been made to develop new therapeutic approaches.
Enzyme replacement therapy with
hPAH could be regarded as a potential form of PKU treatment if the reported in vitro
hPAH instability could be overcome. In this study, we investigated the effect of different
polyol compounds, e.g.
glycerol,
mannitol and PEG-6000 on the in vitro stability of purified
hPAH produced in a heterologous prokaryotic expression system. The recombinant human
enzyme was stored in the presence of the studied
stabilizing agents at different temperatures (4 and -20 degrees C) during a 1-month period.
Protein content, degradation products, specific activity, oligomeric profile and conformational characteristics were assessed during storage. The obtained results showed that the use of 50%
glycerol or 10%
mannitol, at -20 degrees C, protected the
enzyme from loss of its enzymatic activity. The determined DeltaG(0) and quenching parameters indicate the occurrence of conformational changes, which may be responsible for the observed increase in catalytic efficiency.