Difference between revisions of "Fossbakk 2005 Anal Biochem"
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Revision as of 15:57, 17 November 2011
| Fossbakk A, Haavik J (2005) An oxygraphic method for determining kinetic properties and catalytic mechanism of aromatic amino acid hydroxylases. Anal. Biochem. 343: 100-105. |
Fossbakk A, Haavik J (2005) Analyt. Biochem.
Abstract: 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 H2O2 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. • Keywords: Oxygen electrode, Tetrahydrobiopterin, Hydrogen peroxide, Phenylalanine hydroxylase, Tyrosine hydroxylase, Tryptophan hydroxylase, Phenylketonuria
Labels:
Stress:Hypoxia Organism: Human
Preparation: Oxidase; Biochemical Oxidation"Oxidase; Biochemical Oxidation" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property., Enzyme
HRR: Oxygraph-2k
