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 use d to identify structurally and functionally critical regions of recomb inant wild-type human phenylalanine hydroxylase (wt-hPAH; Ser2-Lys452) . The wild-type form consisted of dimeric and tetrameric forms in equi librium, and only the isolated tetrameric form showed positive coopera tivity of substrate (L-Phe) binding (Hill coefficient h = 2.2, S-0.5 = 154 mu M). 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 carbo xy terminus thus seems to contain a motif required for dimer-dimer int eraction in wt-hPAH. The deletion mutants hPAH(Asp112-Lys452), hPAH(Se r2-Gln428) and hPAH(Gly103-Gln428) were all activated by prior incubat ion with L-Phe, but did not reveal any positive cooperativity of subst rate binding (h = 1.0). The activation by L-Phe was accompanied by a m easurable conformational change (as probed by intrinsic fluorescence s pectroscopy) only in the enzyme forms containing the amino-terminal se quence, i.e. wt-hPAH and the Ser2-Gln428 mutant. The amino-terminal de letion mutants hPAH(Asp112-Lys452) and hPAH(Gly103-Gln428) revealed hi gh specific activity, increased apparent affinity for L-Phe (S-0.5 = 6 0 mu M) and a tryptophan fluorescence emission spectrum similar to tha t of the L-Phe-activated wt-hPAH. Moreover, prior incubation of the en zyme forms with lysophosphatidylcholine, a commonly used activator of the PAH, only increased the activity of those forms containing the wt- hPAH aminoterminal sequence. Our results are compatible with a model i n which incubation of wt-hPAH with L-Phe induces both a conformational change (with cooperativity in the tetrameric enzyme) which relieves t he inhibition imposed by the amino-terminal domain to the high-affinit y binding of L-Phe, and an additional activation, as observed for the truncated forms lacking the amino-terminal.