title: Use of surface plasmon resonance for real-time measurements of the global conformational transition in human phenylalanine hydroxylase in response to substrate binding and catalytic activation.
abstract: In the present study the optical biosensor technique, based on the surface plasmon resonance (SPR) phenomenon, was used for real-time measurements of the reversible binding of the pterin cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) and l-phenylalanine (l-Phe) to human phenylalanine hydroxylase (hPAH). When BH(4) (241 Da) was injected over the sensor chip with immobilized tetrameric wt-hPAH a positive DeltaRU response was observed with a square-wave type of sensorgram and a saturable response (about 25 RU/(pmol subunit/mm(2)) with a [S](0.5) value of 5.6 +/- 0.8 microM for the pterin cofactor. The rapid on-and-off rates were, however, not possible to determine. By contrast, when l-Phe (165 Da) was injected a time-dependent increase in RU (up to about 3 min) and a much higher saturable DeltaRU response (about 75 RU/(pmol subunit/mm(2)) at 2 mM l-Phe) than expected (i.e., <5 RU/(pmol subunit/mm(2))) from the low molecular mass of l-Phe were observed in the sensorgram. The half-time for the on-and-off rates were 6 +/- 2 and 9 +/- 1 s, respectively, at 2 mM l-Phe. The steady-state (apparent equilibrium) response revealed a hyperbolic concentration dependence with a [S](0.5) value of 98 +/- 7 microM. The [S](0.5) values of both pterin cofactor and l-Phe were lower than those determined by steady-state enzyme kinetic analysis. Evidence is presented that the DeltaRU response to l-Phe is accounted for by the global conformational transition which occurs in the enzyme upon l-Phe binding, i.e., by the slow reversible transition from a low activity state ("T"-state) to a high activity state ("R"-state) characteristic of this hysteretic enzyme.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Bergen, Bergen, N-5009, Norway. torgeir.flatmark@pki.uib.no
authors.forename: T
authors.initials: T
authors.lastname: Flatmark
authors.forename: A J
authors.initials: AJ
authors.lastname: Stokka
authors.forename: S V
authors.initials: SV
authors.lastname: Berge
mesh_terms: D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D002384:Catalysis; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D010649:Phenylalanine / Q000378:metabolism; D010651:Phenylalanine Hydroxylase / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D001708:Biopterins; D010649:Phenylalanine; D010651:Phenylalanine Hydroxylase; C003402:sapropterin