| Classical phenylketonuria (PKU) is the most common inherited disorder of amino acid metabolism, with an incidence of 1 in 10,000 Caucasians. In 99% of cases the cause of this disease is the dysfunction of the liver enzyme phenylalanine hydroxylase (PAH) which, in affected individuals, fails to clear the body of excess phenylalanine, allowing the amino acid to build up to toxic levels. Here we present the successful expression, purification and characterization of three mutants of (PAH) that cause PKU: Arg158Gln Glu280Lys and Val388Met.; All three mutants were found to be structurally identical to wt (PAH) based on optical spectroscopy and gel-exclusion chromatography. They all had iron occupancy of levels comparable with wild type PAR Furthermore the iron centers in each protein; were shown by optical spectroscopy and EPR to be identical to wt (PAH).; The differences between wt (PAH) and Arg158Gln were identified as uncoupling of oxidation of pterin from hydroxylation of substrate and other minor differences in the kinetic parameters, including a shift in pH optimum from pH 7.0 for wt (PAH) to pH 6.4 for R158Q, as well as substrate inhibition at high levels of L-Phenylalanine. All three mutants suffer a loss of positive cooperativity in response to the substrate.; Comparison between wt (PAH) and Val388Met, revealed differences that are less obvious and harder to define. There is a slight uncoupling (25%) of the reaction and Kmphe is approximately twice that of wt PAR Furthermore, gel-filtration studies indicate a strong propensity for dimer formation at pH 6.8, and preliminary pH dependence studies of the kinetics implicate a cysteine residue as the ionizing residue in the mechanism of this enzyme. Based on these results, our hypothesis is that the mutation of valine to methionine at position 388 causes minor structural changes.; The third mutant, Glu280Lys has been less characterized than either of the other two mutants due to its very low activity, which at 0.09 units/mg is less than 1% of wt activity. The reaction is almost completely uncoupled (<5%). The mutation lies in the putative pterin binding site. However, contrary to expectations, this mutant appears to interact with the cofactor in the same way as does wt (PAH) as shown by EPR and fluorescence studies using the redox inactive cofactor 5-deaza-6-methylpterin. The difference in interaction may be one of orientation rather than the ability to bind. This will be probed by more sensitive pulsed EPR techniques. (Abstract shortened by UMI.)... |
