Pm. Knappskog et A. Martinez, EFFECT OF MUTATIONS AT CYS(237) ON THE ACTIVATION STATE AND ACTIVITY OF HUMAN PHENYLALANINE-HYDROXYLASE, FEBS letters, 409(1), 1997, pp. 7-11
Wild-type recombinant human phenylalanine hydroxylase (wt-hPAH) is act
ivated about 1.5-fold by exposure to alkaline pH (pH 8.5-9.0). In orde
r to study whereas this activation might be related to the activation
of the rat enzyme by N-ethylamaleimide-modification of Cys(237) [Gibbs
and Benkovic (1991) Biochemistry 30, 6795], mutant proteins of hPAH w
ith Cys(237) changed to Ser (S) or Glu (D) have been prepared, The mut
ant forms have high specific activity at pH 7.0 and high affinity for
L-Phe, notably for hPAH-C237D, which shows a 3-fold higher activity th
an L-Phe-activated wt-hPAH and it is not further activated by pre-incu
bation with L-Phe, Moreover, the emission maximum of the intrinsic flu
orescence of hPAH-C237D (lambda(maxem) = 347 nm) resembles that of act
ivated forms of wt-hPAH, However, the activity of this mutant at neutr
al pH is further activated by exposure to alkaline pH, indicating that
activation of wt-hPAH by alkaline pH is not restricted to ionization
of Cys(237). (C) 1997 Federation of European Biochemical Societies.