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Effect of mutations at Cys 237 on the activation state and activity of human phenylalanine hydroxylase
Author(s) -
Knappskog Per M.,
Martı́nez Aurora
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(97)00465-1
Subject(s) - chemistry , phenylalanine hydroxylase , phenylalanine , kinetics , enzyme , mutant , site directed mutagenesis , nuclear chemistry , biochemistry , amino acid , physics , quantum mechanics , gene
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 Cys 237 [Gibbs and Benkovic (1991) Biochemistry 30, 6795], mutant proteins of hPAH with Cys 237 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 (λ max em =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 Cys 237 .