Structure/function relationships in human phenylalanine hydroxylase. Effect of terminal deletions on the oligomerization, activation and cooperativity of substrate binding to the enzyme.
Amino-terminal and carboxy-terminal deletion mutagenesis have been used to identify structurally and functionally critical regions of recombinant wild-type human phenylalanine hydroxylase (wt-hPAH; Ser2-Lys452). The wild-type form consisted of dimeric and tetrameric forms in equilibrium, and only the isolated tetrameric form showed positive cooperativity of substrate (L-Phe) binding (Hill coefficient h = 2.2, S0.5 = 154 microM). The deletion mutants lacking the carboxy-terminal 24 amino acids hPAH (Ser2-Gln428) and hPAH(Gly103-Gln428) formed catalytically active dimers, and incubation with L-Phe did not promote the formation of tetramers, a characteristic property of dimeric wt-hPAH. The carboxyterminus thus seems to contain a motif required for dimer-dimer interaction in wt-hPAH. The deletion mutants hPAH(Asp112-Lys452), hPAH(Ser2-Gln428) and hPAH(Gly103-Gln428) were all activated by prior incubation with L-Phe, but did not reveal any positive cooperativity of substrate binding (h = 1.0). The activation by L-Phe was accompanied by a measurable conformational change (as probed by intrinsic fluorescence spectroscopy) only in the enzyme forms containing the amino-terminal sequence. i.e. wt-hPAH and the Ser2-Gln428 mutant. The amino-terminal deletion mutants hPAH(Asp112-Lys452) and hPAH(Gly103-Gln428) revealed high specific activity, increased apparent affinity for L-Phe (S0.5 = 60 microM) and a tryptophan fluorescence emission spectrum similar to that of the L-Phe-activated wt-hPAH. Moreover, prior incubation of the enzyme forms with lysophosphatidylcholine, a commonly used activator of the PAH, only increased the activity of those forms containing the wt-hPAH amino-terminal sequence. Our results are compatible with a model in which incubation of wt-hPAH with L-Phe induces both a conformational change (with cooperativity in the tetrameric enzyme) which relieves the inhibition imposed by the amino-terminal domain to the high-affinity binding of L-Phe, and an additional activation, as observed for the truncated forms lacking the amino-terminal.
Knappskog
P M
PM
Department of Biochemistry and Molecular Biology, University of Bergen, Norway.
Flatmark
T
T
Aarden
J M
JM
Haavik
J
J
Martínez
A
A
D000494:Allosteric Regulation; D004789:Enzyme Activation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000378:metabolism; D010651:Phenylalanine Hydroxylase / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D008244:Lysophosphatidylcholines; D011993:Recombinant Fusion Proteins; D010649:Phenylalanine; D007501:Iron; D010651:Phenylalanine Hydroxylase