Structure/Function Relationships in Human Phenylalanine Hydroxylase

Abstract

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, S 0.5 = 154 μM). 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 carboxy‐terminus 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(Glyl03‐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 (S 0.5 = 60 μM) 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.