THE ROLE OF PHENYLALANINE IN STRUCTURE-FUNCTION-RELATIONSHIPS OF PHENYLALANINE-HYDROXYLASE REVEALED BY RADIATION TARGET ANALYSIS

The activity of rat liver phenylalanine hydroxylase (PAH; phenylalanin e 4-monooxygenase, EC 1.14.16.1) is regulated by interaction with its substrate, phenylalanine, and its coenzyme, BH4 [tetrahydrobiopterin y droxypropyl-L-erythro-5,6,7,8-tetrahydropterin)]. The structural chang es accompanying these interactions have been studied by radiation targ et analysis, PAH purified from rat liver was incubated with 2 mM pheny lalanine to achieve complete activation of the enzyme, Frozen samples were irradiated with various doses of high energy electrons; samples w ere subsequently thawed, and several surviving properties of the enzym e were determined. Each parameter decreased as a single exponential fu nction of radiation dose, Radiation target analysis of enzymatic activ ity yielded a dimeric target size, Similar radiation effects on subuni t monomers and on tetrameric structure were observed. Together with re sults from unactivated enzyme, these data show that phenylalanine incr eases 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.