The role of phenylalanine in structure-function relationships of phenylalanine hydroxylase revealed by radiation target analysis.
The activity of rat liver phenylalanine hydroxylase (PAH; phenylalanine 4-monooxygenase, EC 1.14.16.1) is regulated by interaction with its substrate, phenylalanine, and its coenzyme, BH4 [tetrahydrobiopterin (6R-dihydroxypropyl-L-erythro-5,6,7,8-tetrahydropterin)]. The structural changes accompanying these interactions have been studied by radiation target analysis. PAH purified from rat liver was incubated with 2 mM phenylalanine to achieve complete activation of the enzyme. Frozen samples were irradiated with various doses of high energy electrons; samples were subsequently thawed, and several surviving properties of the enzyme were determined. Each parameter decreased as a single exponential function of radiation dose. Radiation target analysis of enzymatic activity yielded a dimeric target size. Similar radiation effects on subunit monomers and on tetrameric structure were observed. Together with results from unactivated enzyme, these data show that phenylalanine increases the interactions between the subunits in a dimer and weakens the interactions between dimers in a tetramer. These alterations prevent the natural cofactor, a tetrahydrobiopterin, from exerting a negative effect on activity.
Davis
M D
MD
National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, USA.
Parniak
M A
MA
Kaufman
S
S
Kempner
E
E
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004307:Dose-Response Relationship, Radiation; D004789:Enzyme Activation; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D010649:Phenylalanine / Q000737:chemistry*; D010651:Phenylalanine Hydroxylase / Q000737:chemistry* / Q000528:radiation effects; D051381:Rats
D046911:Macromolecular Substances; D001708:Biopterins; D010649:Phenylalanine; D010651:Phenylalanine Hydroxylase; C003402:sapropterin