Related Experiment Videos
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
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.
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.
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.