Effect of mutations at Cys237 on the activation state and activity of human phenylalanine hydroxylase

FEBS Letters

Knappskog, Per M.¹  Martı́nez, Aurora¹ 

[1] Department of Biochemistry and Molecular Biology, University of Bergen, Årstadveien 19, N-5009 Bergen, Norway

关键词: Phenylalanine hydroxylase;    Site-directed mutagenesis;    Enzyme activation;    Cooperativity;    Tryptophan fluorescence;    BH4;    (6R)-l-erythro-tetrahydrobiopterin;    NEM;    N-ethylmaleimide;    PAH;    phenylalanine hydroxylase;    hPAH;    human phenylalanine hydroxylase;    rPAH;    rat phenylalanine hydroxylase;    MBP;    maltose-binding protein;    PKU;    phenylketonuria;    wt-hPAH;    wild-type human phenylalanine hydroxylase;   

DOI  :  10.1016/S0014-5793(97)00465-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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, 6795], 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 (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 Cys²³⁷.

【 授权许可】

Unknown