Tyrosine Hydroxylase (TyrH) is a pterin-dependent mononuclear non-heme iron
oxygenase. TyrH catalyzes the hydroxylation reaction of tyrosine to
dihydroxyphenylalanine (DOPA). This reaction is the first and the rate-limiting step in
the biosynthesis of the catecholamine neurotransmitters. The active site iron in TyrH is
coordinated by the common facial triad motif, 2-His-1-Glu. A combination of kinetic
and spectroscopic techniques was applied in order to obtain insight into the catalytic
mechanism of this physiologically important enzyme.
Analysis of the TyrH reaction by rapid freeze-quench Mossbauer spectroscopy
allowed the first direct characterization of an Fe(IV) intermediate in a mononuclear nonheme
enzyme catalyzing aromatic hydroxylation. Further rapid kinetic studies
established the kinetic competency of this intermediate to be the long-postulated
hydroxylating species, Fe(IV)O.
Spectroscopic investigations of wild-type (WT) and mutant TyrH complexes
using magnetic circular dichroism (MCD) and X-ray absorption spectroscopy (XAS)
showed that the active site iron is 6-coordinate in the resting form of the enzyme and that binding of either tyrosine or 6MPH4 alone does not change the coordination. However,
when both tyrosine and 6MPH4 are bound, the active site becomes 5-coordinate, creating
an open site for reaction with O2. Investigation of the kinetics of oxygen reactivity of
TyrH complexes in the absence and presence of tyrosine and/or 6MPH4 indicated that
there is a significant enhancement in reactivity in the 5-coordinate complex in
comparison to the 6-coordinate form. Similar investigations with E332A TyrH showed
that Glu332 residue plays a role in directing the protonation of the bridged complex that
forms prior to the formation of Fe(IV)O.
Rapid chemical quench analyses of DOPA formation showed a burst of product
formation, suggesting a slow product release step. Steady-state viscosity experiments
established a diffusional step as being significantly rate-limiting. Further studies with
stopped-flow spectroscopy indicated that the rate of TyrH reaction is determined by a
combination of a number of physical and chemical steps.
Investigation of the NO complexes of TyrH by means of optical absorption,
electron paramagnetic resonance (EPR) and electron spin echo envelope modulation
(ESEEM) techniques revealed the relative positions of the substrate and cofactor with
respect to NO, an O2 mimic, and provided further insight into how the active site is
tuned for catalytic reactivity upon substrate and cofactor binding.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-2009-12-7355 |
| Date | 2009 December 1900 |
| Creators | Eser, Bekir Engin |
| Contributors | Fitzpatrick, Paul F., Raushel, Frank M. |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | application/pdf |
Page generated in 0.0023 seconds