Studies on the regulatory properties of the pterin cofactor and dopamine bound at the active site of human phenylalanine hydroxylase.
The catalytic activity of phenylalanine hydroxylase (PAH, phenylalanine 4-monooxygenase EC 1.14.16.1) is regulated by three main mechanisms, i.e. substrate (l-phenylalanine, L-Phe) activation, pterin cofactor inhibition and phosphorylation of a single serine (Ser16) residue. To address the molecular basis for the inhibition by the natural cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin, its effects on the recombinant tetrameric human enzyme (wt-hPAH) was studied using three different conformational probes, i.e. the limited proteolysis by trypsin, the reversible global conformational transition (hysteresis) triggered by L-Phe binding, as measured in real time by surface plasmon resonance analysis, and the rate of phosphorylation of Ser16 by cAMP-dependent protein kinase. Comparison of the inhibitory properties of the natural cofactor with the available three-dimensional crystal structure information on the ligand-free, the binary and the ternary complexes, have provided important clues concerning the molecular mechanism for the negative modulatory effects. In the binary complex, the binding of the cofactor at the active site results in the formation of stabilizing hydrogen bonds between the dihydroxypropyl side-chain and the carbonyl oxygen of Ser23 in the autoregulatory sequence. L-Phe binding triggers local as well as global conformational changes of the protomer resulting in a displacement of the cofactor bound at the active site by 2.6 A (mean distance) in the direction of the iron and Glu286 which causes a loss of the stabilizing hydrogen bonds present in the binary complex and thereby a complete reversal of the pterin cofactor as a negative effector. The negative modulatory properties of the inhibitor dopamine, bound by bidentate coordination to the active site iron, is explained by a similar molecular mechanism including its reversal by substrate binding. Although the pterin cofactor and the substrate bind at distinctly different sites, the local conformational changes imposed by their binding at the active site have a mutual effect on their respective binding affinities.
Solstad
Therese
T
Department of Biochemistry and Molecular Biology, University of Bergen, Arstadveien 19, N-5009 Bergen, Norway.
Stokka
Anne J
AJ
Andersen
Ole A
OA
Flatmark
Torgeir
T
D001665:Binding Sites; D020134:Catalytic Domain; D003067:Coenzymes; D004298:Dopamine / Q000378:metabolism*; D006801:Humans; D008667:Metalloproteins / Q000378:metabolism*; D000090006:Molybdenum Cofactors; D010651:Phenylalanine Hydroxylase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011621:Pteridines / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020349:Surface Plasmon Resonance
D003067:Coenzymes; D004298:Dopamine; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D010651:Phenylalanine Hydroxylase; D011621:Pteridines; D011994:Recombinant Proteins; C022261:molybdopterin