Biophysical and kinetic studies on wild type and point mutants of phenylalanine hydroxylase from Chromobacterium violaceum

Phenylalanine hydroxylase (PAH) is an enzyme that catalyzes the conversion of the amino acid phenylalanine to tyrosine. Individuals who lack a functional PAH have high phenylalanine blood levels that cause brain damage and lead to mental retardation. This metabolic disorder is called phenylketonuria (PKU) and one out of ten thousand newborns has a defective PAH and is in danger of becoming mentally retarded. Nowadays, our defense against PKU consists of an early diagnosis and a special diet. The main component of the phenylalanine-restricted diet is a jelly offensive in odor and taste so it is not surprising that many patients fail to comply and develop mental retardation. An alternative approach to treating PKU is enzyme replacement therapy. PAH is the ideal candidate for enzyme replacement therapy of PKU because it can reduce phenylalanine level in the blood. Since the human PAH is unstable, a bacterial PAH from Chromobacterium violaceum (cPAH) was chosen. Before initiating clinical trials it is necessary to thoroughly characterize the enzyme. The effect of metals, temperature and pH on the catalytic activity and stability of cPAH was studied by enzyme assays, circular Dichroism, and differential scanning calorimetry. Iron(II) is necessary for catalytic activity and enhances the resistance of cPAH to thermal denaturation, whereas Co(II) inhibits the enzyme and increases its stability. The optimum pH for stability is 7.4. In addition, the catalytic properties and stability of two point mutants, Y179A and Y179F, were studied. The results showed that Y179 is important for catalysis and binding of the substrate phenylalanine. In conclusion, cPAH is a robust catalyst that could be used in treating PKU patients.