EFFECT OF MUTATIONS AT CYS(237) ON THE ACTIVATION STATE AND ACTIVITY OF HUMAN PHENYLALANINE-HYDROXYLASE

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.