Polyol additives modulate the in vitro stability and activity of recombinant human phenylalanine hydroxylase.
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.
Nascimento
Cátia
C
Metabolism and Genetics Group, iMed.UL, Faculdade Farmácia da Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
Leandro
João
J
Lino
Paulo Roque
PR
Ramos
Luís
L
Almeida
António José
AJ
de Almeida
Isabel Tavares
IT
Leandro
Paula
P
D055162:Biocatalysis / Q000187:drug effects; D002850:Chromatography, Gel; D004795:Enzyme Stability / Q000187:drug effects; D006801:Humans; D010651:Phenylalanine Hydroxylase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011108:Polymers / Q000494:pharmacology*; D011489:Protein Denaturation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014508:Urea / Q000494:pharmacology
D011108:Polymers; D011994:Recombinant Proteins; C024617:polyol; D014508:Urea; D010651:Phenylalanine Hydroxylase