Fossbakk 2016 Dissertation

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Fossbakk A (2016) Human tyrosine hydroxylase: Oxygen dependence and role in Dopa responsive dystonia. Dissertation p60.

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Fossbakk A (2016) Dissertation

Abstract: The aromatic amino acid hydroxylases (AAHs); phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH), tryptophan hydroxylase 1 and 2 (TPH 1/TPH 2); are structurally and functionally related enzymes. All AAHs require iron, dioxygen and the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) to be catalytically active. PAH is the first and rate-limiting enzyme in the catabolism of L-Phe; TH is the ratelimiting enzyme in the synthesis of the catecholamine neurotransmitters dopamine, adrenaline and noradrenaline; while the TPH 1 and 2 catalyses the first and ratelimiting reaction in then synthesis of serotonin and melatonin.

These physiologically important enzymes are implicated in human diseases; polymorphisms and variants in the TPH genes are associated to neuropsychiatric disorders, mutations in PAH and TH are responsible for the autosomal recessive disorders phenylketonuria (PKU) and TH deficiency (THD), respectively. Furthermore, a role of TH in Parkinson’s disease and hypoxia induced diseases, such as altitude sickness and sleep apnoea, has been suggested.

The aims of this project were to 1) develop improved methods of studying AAHs, mainly focusing on their dependence of oxygen; 2) investigate the role of oxygen in the AAH reactions, with an emphasis on TH; 3) to characterize the effect of mutations in AAH, mainly PAH and TH, on their enzyme function and stability; 4) investigate the effects of missense TH mutations reported in patients with THD and perform genotype-phenotype comparisons in these patients.

O2k-Network Lab: NO Bergen Tronstad KJ

Labels: MiParea: Respiration 

Organism: Human 

Preparation: Enzyme, Oxidase;biochemical oxidation 

Regulation: Oxygen kinetics 

HRR: Oxygraph-2k 


Abstract continued

The thesis is based on three separate articles (Article 1-3); the first article is focused on developing a new oxygrapic assay method for studying the activity and enzymatic mechanism of AAHs by monitoring the oxygen consumption continuously during the catalytic reaction. The second paper describes the oxygen dependence of TH in normoxic and hypoxic conditions, relevant for physiological effects of high altitude and other conditions of low oxygen availability. In the third article, mutated variants of TH reported in patients with THD, were characterized and compared to wild-type (wt)-TH with regards to in vitro solubility, thermal stability and kinetic properties.

In Article 1 we demonstrated the utility of a new oxygraphic assay to study the function of AAHs. We studied kinetic properties and enzyme reaction mechanisms of both wt and mutant enzyme using different substrates and cofactors. A stable reaction stoichiometry of 1:1 was obtained between the amount of oxygen consumed and tyrosine formation when the natural cofactor (6R)-tetrahydrobiopterin was added as electron donor in the phenylalanine hydroxylase (PAH) reaction. In comparison, low and variable coupling efficiency values between oxygen consumption and tyrosine formation were found using the parent unsubstituted tetrahydropterin. Furthermore, we studied the phenylketonuria-associated PAH mutant R158Q and found that the reaction had a coupling efficiency of about 80 % compared to the wild-type enzyme under similar conditions. The high time resolution of this method allowed us to obtain new knowledge about the initial reaction kinetics of the AAHs.

These findings were investigated further in Article 2; where we observed an initial high activity phase in the first 1-2 minutes of the TH reaction, levelling off to a lower stable activity rate after the initial phase. During the initial reaction phase, apparent Km-values of 29–45 μM for dioxygen were determined for all human TH isoforms, i.e. 2–40 times higher than previously reported for TH isolated from animal tissues. After 8 min incubation, the Km (O2)-values had declined to an average of 20 ± 4 μM.

In Article 3, 22 different missense and one nonsense coding variants from patients with THD were produced in E. coli and subjected to biochemical studies of their enzymatic properties. Compared to wt-TH we observed a great heterogeneity of changes in the in vitro solubility, thermal stability and enzymatic activity among the mutated TH variants; indicating different pathogenetic mechanisms of the TH mutations found in patients with THD.

In conclusion, this project has established the new oxygraphic method as a valuable supplement to other activity assays of AAHs, providing an assay which is versatile, fairly sensitive and has a high time resolution. New insights into the initial phase of the enzymatic reaction of the AAHs has revealed a previously undescribed shift to a low activity phase of the enzymes in vitro, which may be due to a rate limiting regeneration of the active site iron from the inactive ferric form in the catalytic cycle. This may be significant in understanding the physiological effect of hypoxic conditions, as the concentration of oxygen in tissues reaches the Km-values for TH calculated in this study. Characterization of THD associated variants of mutated TH have increased the understanding of the molecular mechanisms of their pathogenicity; contributing to the understanding of the neurological symptoms and complementing the animal and clinical studies conducted to develop new and personalized treatments for THD patients.