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Structure-function relationships of phenylalanine hydroxylase revealed by radiation target analysis
M D Davis1, M A Parniak, S Kaufman
1National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, USA.
Radiation target analysis reveals phenylalanine hydroxylase (PAH) tetramers contain two active dimers. Tetrahydrobiopterin (BH4) binding to PAH tetramers inhibits enzymatic activity, affecting phenylalanine metabolism.
Area of Science:
- Biochemistry
- Enzymology
- Protein structure
Background:
- Phenylalanine hydroxylase (PAH) is a tetrameric enzyme crucial for phenylalanine metabolism.
- Understanding PAH's subunit organization is key to its function and regulation.
Purpose of the Study:
- To investigate the subunit organization of purified rat liver phenylalanine hydroxylase (PAH).
- To elucidate the role of cofactors, specifically tetrahydrobiopterin (BH4), in PAH activity and structure.
Main Methods:
- Radiation target analysis was employed to determine the functional size of PAH under different cofactor conditions.
- High-performance size-exclusion chromatography (HPSEC) was used to analyze the structural integrity of PAH after irradiation.
Main Results:
- Radiation inactivation yielded a target size of ~120 kDa with 6-methyltetrahydropterin, suggesting a dimer.
- With BH4, inactivation curves were complex, indicating an initial increase then decrease in activity, with a target size of ~100 kDa at high doses.
- HPSEC showed tetramer loss with a target size of 110 kDa.
- Irradiation led to enzyme activation and desensitization to phenylalanine.
Conclusions:
- Purified PAH tetramers are composed of two enzymatically active dimers.
- Tetrahydrobiopterin (BH4) interacts with the PAH tetramer, potentially inhibiting or deactivating enzymatic activity.
- The complex inactivation profile with BH4 suggests a regulatory role for the cofactor at the tetramer level.
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