Effect of mutations at Cys237 on the activation state and activity of human phenylalanine hydroxylase.
Wild-type recombinant human phenylalanine hydroxylase (wt-hPAH) is activated about 1.5-fold by exposure to alkaline pH (pH 8.5-9.0). In order to study whereas this activation might be related to the activation of the rat enzyme by N-ethylamaleimide-modification of Cys237 [Gibbs and Benkovic (1991) Biochemistry 30, 67951, mutant proteins of hPAH with Cys237 changed to Ser (S) or Glu (D) have been prepared. The mutant 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 than L-Phe-activated wt-hPAH and it is not further activated by pre-incubation with L-Phe. Moreover, the emission maximum of the intrinsic fluorescence of hPAH-C237D (lambda(maxem) = 347 nm) resembles that of activated forms of wt-hPAH. However, the activity of this mutant at neutral pH is further activated by exposure to alkaline pH, indicating that activation of wt-hPAH by alkaline pH is not restricted to ionization of Cys237.
Knappskog
P M
PM
Department of Biochemistry and Molecular Biology, University of Bergen, Haukeland Hospital, Norway.
Martínez
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A
D001224:Aspartic Acid / Q000235:genetics; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003545:Cysteine / Q000235:genetics*; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010651:Phenylalanine Hydroxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012694:Serine / Q000235:genetics; D013050:Spectrometry, Fluorescence
D001224:Aspartic Acid; D012694:Serine; D010651:Phenylalanine Hydroxylase; D003545:Cysteine