A comparison of kinetic and regulatory properties of the tetrameric and dimeric forms of wild-type and Thr427 -> Pro mutant human phenylalanine hydroxylase - Contribution of the flexible hinge region Asp425-Gln429 to the tetramerization and cooperative substrate binding

Recombinant human phenylalanine hydroxylase (hPAH, phenylalanine 4-monooxyg enase EC 1.14.16.1) is catalytically active both as a tetramer and a dimer [Knappskog, P.M., Flatmark, T., Aarden, J.M., Haavik, J. and Martinet, A. ( 1996) Eur. J. Biochem. 242, 813-821]. In the present study we have further characterized the differences in kinetic and regulatory properties of the t wo oligomeric forms when expressed in Escherichia coli. The positive cooper ativity of L-Phe binding to the tetrameric form both in enzyme kinetic stud ies (h = 1.6) and intrinsic tryptophan fluorescence measurements (h = 2.3) was abolished in the dimer, which also revealed a catalytic efficiency (V-m ax/[S](0.5)) of only 35% of the tetramer. Whereas the catalytic activity of the tetramer was activated fivefold to sixfold by preincubation with L-Phe , the dimer revealed only a 1.6-fold activation. The crystal structure has identified a five-residue flexible hinge region (Asp425-Gln429) that links the beta -strand T beta2 (Ile421-Leu424) and the 24 residue amphipathic alp ha -helix Tal (Gln428-Lys452) at the C-terminus which forms an antiparallel colied-coli structure in the center of the tetramer [Fusetti, F. Erlandsen , H., Flatmark, T. & Stevens, R.C. (1998) J. Biol. Chem. 273, 16962-16967]. The potential role of this flexible hinge in the tetramerization and the c onformational transition of wt-hPAH ion the cooperative binding of L-Phe wa s examined by site-specific mutagenesis. Substitution of Thr427 by a Pro (a s in tyrosine hydroxylase) resulted in a mutant protein which was isolated mainly (about 95%) as a dimer. The isolated tetramer of T427P revealed no k inetic cooperativity of L-Phe binding, the catalytic efficiency (V-max/[S]( 0.5)) was decreased to about 39% of the wild-type tetramer and it was not a ctivated by L-Phe preincubation. The dimeric forms of T427P and wt-hPAH rev ealed rather similar kinetic properties. The lack of kinetic cooperativity of the T427P tetramer was associated with a corresponding change in the bin ding isotherm for L-Phe as studied by intrinsic tryptophan fluorescence mea surements. Protein stability was also reduced both for the E. coli expresse d and the in vitro synthesized mutant enzyme. Collectively, these results i ndicate that the positive cooperativity of L-Phe binding to wt-hPAH require s a tetrameric enzyme with a C-terminal flexibel hinge region (Asp425-Gln42 9) which has a structural role in the formation of the enzyme tetramer. Fur thermore, this hinge region represents a motif in the PAH structure that is involved in the conformational change transmitted through the protein on t he cooperative binding of L-Phe to tetrameric wt-hPAH. This conclusion is f urther supported by studies on two disease (phenylketonuria)-associated mut ant forms.