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Substrate-assisted catalysis in cytochrome P450eryF

J R Cupp-Vickery1, O Han, C R Hutchinson

  • 1Department of Molecular Biology and Biochemistry, University of California, Irvine 92717, USA.

Nature Structural Biology
|July 1, 1996
PubMed
Summary

Cytochrome P450eryF uses a unique active site alanine and a water molecule to cleave dioxygen. Mutating these or altering the substrate significantly reduced catalytic activity, revealing a novel substrate-assisted catalytic mechanism.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • Cytochromes P450 are crucial enzymes in biological systems.
  • A conserved active site threonine is typically involved in O2 binding and cleavage.
  • Cytochrome P450eryF presents an atypical active site with alanine and an ordered water molecule (Wat 519).

Purpose of the Study:

  • To investigate the roles of the active site alanine and Wat 519 in Cytochrome P450eryF catalysis.
  • To understand the mechanism of dioxygen bond cleavage in this unusual P450 enzyme.

Main Methods:

  • Site-directed mutagenesis to create Ala --> Ser and Ala --> Thr variants of P450eryF.
  • Enzymatic assays using a substrate analogue lacking a 5-hydroxyl group.
  • Kinetic, spectral, and crystallographic analyses of wild-type and mutant enzymes.

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Main Results:

  • Mutant enzymes exhibited decreased catalytic activity compared to wild-type P450eryF.
  • The catalytic activity loss correlated with the disappearance or altered positioning of Wat 519.
  • Substrate analogue studies further supported the importance of the 5-hydroxyl group and Wat 519.

Conclusions:

  • The alanine residue and Wat 519 play critical roles in the catalytic activity of P450eryF.
  • P450eryF employs a substrate-assisted mechanism for acid-catalyzed dioxygen bond cleavage.
  • This study reveals a novel catalytic strategy in the cytochrome P450 superfamily.