© 2006 The Japanese Biochemical Society.
Regular Paper |
Molecular Mechanism for Pterin-Mediated Inactivation of Tyrosine Hydroxylase: Formation of Insoluble Aggregates of Tyrosine Hydroxylase
1 Department of Life Science and 3 Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501; 2 Institute for Comprehensive Medical Science and 4 Department of Hygiene, School of Medicine, Fujita Health University, Toyoake 470-1192; 5 Graduate School of Environmental Studies, Nagoya University, Nagoya 464-8601; and 6 Japan Association for the Advancement of Medical Equipment, Tokyo 113-0033
* To whom correspondence should be addressed. Tel: +81-45-924-5822, Fax: +81-45-924-5807, E-mail: hichinos{at}bio.titech.ac.jp
Tyrosine hydroxylase (TH), an iron-containing enzyme, catalyzes the first and rate-limiting step of catecholamine biosynthesis, and requires tetrahydrobiopterin (BH4) as a cofactor. We found that preincubation of recombinant human TH with BH4 results in the irreversible inactivation of the enzyme at a concentration far less than the Km value toward BH4 in spite of its cofactor role, whereas oxidized biopterin, which has no cofactor activity, does not affect the enzyme activity. We show that TH is inactivated by BH4 in competition with the binding of dopamine. The sequential addition of BH4 to TH results in a gradual decrease in the intensity of the fluorescence and CD spectra without changing their overall profiles. Sedimentation velocity analysis demonstrated an association of TH molecules with each other in the presence of BH4, and studies using gel-permeation chromatography, turbidity measurements, and transmission electron microscopy demonstrated the formation of amorphous aggregates with large molecular weights following the association of the TH proteins. These results suggest that BH4 not only acts as a cofactor, but also accelerates the aggregation of TH. We propose a novel mechanism for regulating the amount of TH protein, and discuss its physiological significance.