title: Substrate-induced conformational transition in human phenylalanine hydroxylase as studied by surface plasmon resonance analyses: the effect of terminal deletions, substrate analogues and phosphorylation.
abstract: The optical biosensor technique, based on the surface plasmon resonance (SPR) phenomenon, was used for real-time measurements of the slow conformational transition (isomerization) which occurs in human phenylalanine hydroxylase (hPAH) on the binding/dissociation of L-phenylalanine (L-Phe). The binding to immobilized tetrameric wt-hPAH resulted in a time-dependent increase in the refractive index (up to approx. 3 min at 25 degrees C) with an end point of approx. 75 RU (resonance units)/(pmol subunit/mm(2)). By contrast, the contribution of binding the substrate (165 Da) to its catalytic core enzyme [DeltaN(1-102)/DeltaC(428-452)-hPAH] was only approx. 2 RU/(pmol subunit/mm(2)). The binding isotherm for tetrameric and dimeric wt-hPAH revealed a [S](0.5)-value of 98+/-7 microM (h =1.0) and 158+/-11 microM, respectively, i.e. for the tetramer it is slightly lower than the value (145+/-5 microM) obtained for the co-operative binding (h =1.6+/-0.4) of L-Phe as measured by the change in intrinsic tryptophan fluorescence. The responses obtained by SPR and intrinsic tryptophan fluorescence are both considered to be related to the slow reversible conformational transition which occurs in the enzyme upon L-Phe binding, i.e. by the transition from a low-activity state ('T-state') to a relaxed high-activity state ('R-state') characteristic of this hysteretic enzyme, however, the two methods reflect different elements of the transition. Studies on the N- and C-terminal truncated forms revealed that the N-terminal regulatory domain (residues 1-117) plus catalytic domain (residues 118-411) were required for the full signal amplitude of the SPR response. Both the on- and off-rates for the conformational transition were biphasic, which is interpreted in terms of a difference in the energy barrier and the rate by which the two domains (catalytic and regulatory) undergo a conformational change. The substrate analogue 3-(2-thienyl)-L-alanine revealed an SPR response comparable with that of L-Phe on binding to wild-type hPAH.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Bergen, Arstadveien 19, N-5009 Bergen, Norway.
authors.forename: Anne J
authors.initials: AJ
authors.lastname: Stokka
authors.forename: Torgeir
authors.initials: T
authors.lastname: Flatmark
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000378:metabolism; D020134:Catalytic Domain; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006801:Humans; D010649:Phenylalanine / Q000378:metabolism*; D010651:Phenylalanine Hydroxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D017384:Sequence Deletion; D012694:Serine / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D014364:Tryptophan / Q000737:chemistry
chemical_list: C047942:3-(2-thienyl)alanine; D012694:Serine; D010649:Phenylalanine; D014364:Tryptophan; D010651:Phenylalanine Hydroxylase; D017868:Cyclic AMP-Dependent Protein Kinases; D000409:Alanine