Oxygen dependence of tyrosine hydroxylase

Abstract

The effects of dioxygen on tyrosine hydroxylase (TH) activity was studied, measuring the formation of DOPA from tyrosine, (3)H(2)O from 3,5-(3)H-tyrosine, or by direct oxygraphic determination of oxygen consumption. A high enzyme activity was observed during the initial 1-2 min of the reactions, followed by a decline in activity, possibly related to a turnover dependent substoichiometrical oxidation of enzyme bound Fe(II) to the inactive Fe(III) state. During the initial reaction phase, apparent K (m)-values of 29-45 microM 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 K (m) (O(2))-values had declined to an average of 20 +/- 4 microM. Thus, TH activity may be severely limited by oxygen availability even at moderate hypoxic conditions, and the enzyme is rapidly and turnover dependent inactivated at the experimental conditions commonly employed to measure in vitro activities.

Functional Studies of Tyrosine Hydroxylase Missense Variants Reveal Distinct Patterns of Molecular Defects in Dopa ‐Responsive Dystonia

“…As previously described [Rostrup et al., 2008 ], the wt TH reaction has a characteristic pattern of oxygen consumption with an initial burst phase and a steady-state reaction rate after 1–2 min [Flatmark et al., 1999 ]. This reaction pattern and the time courses for oxygen consumption in the oxygraphic assay and l -Dopa production (measured by HPLC) and coupling ratios were similar for wt TH and the mutant TH variants that had measurable activity (Supp.…”

Polarographic Oxygen Sensors, the Oxygraph, and High‐Resolution Respirometry to Assess Mitochondrial Function

“…Appart from the oxygraphic assay of cytochrome c oxidase activity [7] and activity measurements of other oxygen-consuming enzymes [10], diagnostic substrate-uncouplerinhibitor titration protocols in mitochondrial respiratory studies [11][12][13] yield information on the capacities of metabolic pathways rather than individual enzymes, and thus provide insight into pathological effects on integrated mitochondrial function. It is well established that various defects in oxidative phosphorylation (OXPHOS) can be identified by the measurement of oxygen consumption (respirometric OXPHOS analysis) in fresh sample preparations that are not detected via more focused enzymatic analysis, via measurement of mitochondrial membrane potential, or via electron microscopic analyses of mitochondrial structure [11].…”

Searching for molecular hypoxia sensors among oxygen-dependent enzymes

“…TH catalyzes the hydroxylation of L-tyrosine into L-3,4-dihydroxyphenylalanine (L-DOPA) ( Figure 3H ), the rate-limiting step in biosynthesis of catecholamines (dopamine, noradrenaline, and adrenaline) ( Rostrup et al, 2008 ). The O 2 Km values of TH vary by splice isoform, ranging from 12 to 47 μM across the four splice isoforms as measured in in vitro enzymatic assays ( Table 4 ; Rostrup et al, 2008 ; Katz, 1980 ). In cellular assays, the activity of TH in PC12 cells was inhibited when O 2 concentration decreased from 139 to 33 μM ( Rostrup et al, 2008 ).…”

“…The O 2 Km values of TH vary by splice isoform, ranging from 12 to 47 μM across the four splice isoforms as measured in in vitro enzymatic assays ( Table 4 ; Rostrup et al, 2008 ; Katz, 1980 ). In cellular assays, the activity of TH in PC12 cells was inhibited when O 2 concentration decreased from 139 to 33 μM ( Rostrup et al, 2008 ). These measurements suggest that acute hypoxia inhibiting TH could lead to decreased synthesis of catecholamines ( Figure 2B ; Raghuraman et al, 2012 ; Souvannakitti et al, 2009 ).…”