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.