STRUCTURE FUNCTION RELATIONSHIPS IN HUMAN PHENYLALANINE-HYDROXYLASE -EFFECT OF TERMINAL DELETIONS ON THE OLIGOMERIZATION, ACTIVATION AND COOPERATIVITY OF SUBSTRATE-BINDING TO THE ENZYME/
Pm. Knappskog et al., STRUCTURE FUNCTION RELATIONSHIPS IN HUMAN PHENYLALANINE-HYDROXYLASE -EFFECT OF TERMINAL DELETIONS ON THE OLIGOMERIZATION, ACTIVATION AND COOPERATIVITY OF SUBSTRATE-BINDING TO THE ENZYME/, European journal of biochemistry, 242(3), 1996, pp. 813-821
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.