An oxygraphic method for determining kinetic properties and catalytic mechanism of aromatic amino acid hydroxylases.
We have developed a simple and versatile oxygraphic assay procedure that can be used for determination of kinetic constants and enzyme reaction mechanisms of wild-type and mutant aromatic amino acid hydroxylases. The oxygen concentration and rate of oxygen consumption were measured continuously throughout the enzyme reaction, while aliquots of the reaction mixture were removed at regular intervals for measurement of other substrates and products. Using (6R)-tetrahydrobiopterin as electron donor in the phenylalanine hydroxylase (PAH) reaction, a stable stoichiometry of 1:1 was obtained between the amount of oxygen consumed and the tyrosine formation. In comparison, low and variable coupling efficiency values between oxygen consumption and tyrosine formation were found using the parent unsubstituted tetrahydropterin. The application of this assay procedure to study mechanisms of disease-associated mutations was also demonstrated. Thus, the phenylketonuria-associated PAH mutant R158Q had a coupling efficiency of about 80%, compared to the wild-type enzyme under similar conditions. Furthermore, the amount of H(2)O(2) produced in the reaction catalyzed by R158Q PAH was about four times higher than the amount produced by the wild-type PAH, demonstrating a possible pathogenetic mechanism of the mutant enzyme.
Fossbakk
Agnete
A
Department of Biomedicine, Section of Biochemistry and Molecular Biology, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway.
Haavik
Jan
J
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000737:chemistry; D004566:Electrodes; D006801:Humans; D007700:Kinetics; D010100:Oxygen / Q000032:analysis*; D010651:Phenylalanine Hydroxylase / Q000737:chemistry* / Q000235:genetics; D010661:Phenylketonurias / Q000201:enzymology / Q000235:genetics; D017354:Point Mutation / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry
D001708:Biopterins; D014443:Tyrosine; D010651:Phenylalanine Hydroxylase; C003402:sapropterin; D010100:Oxygen