FEBS Letters | |
Effect of mutations at Cys237 on the activation state and activity of human phenylalanine hydroxylase | |
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 (λmax 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
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