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Catalase-peroxidase active site restructuring by a distant and "inactive" domain
Ruletha D Baker1, Carma O Cook, Douglas C Goodwin
1Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849-5312, USA.
Biochemistry
|June 7, 2006
Summary
The C-terminal domain of catalase-peroxidase (KatG(C)) can restore catalytic activity to the N-terminal domain (KatG(N)). This interaction restructures the heme environment, reactivating both catalase and peroxidase functions.
Area of Science:
- Biochemistry
- Enzymology
- Protein Structure-Function Relationships
Background:
- Catalase-peroxidases possess two distinct domains: an N-terminal heme-binding domain and a non-catalytic C-terminal domain.
- The N-terminal domain alone lacks catalase and peroxidase activity, attributed to distal histidine coordination to the heme iron.
Purpose of the Study:
- To investigate the role of the isolated C-terminal domain (KatG(C)) in restoring the bifunctional activity of the N-terminal domain (KatG(N)).
- To elucidate the structural and functional consequences of interdomain interactions on the catalase-peroxidase active site.
Main Methods:
- Expression and isolation of individual N-terminal (KatG(N)) and C-terminal (KatG(C)) domains.
- Spectroscopic analysis including Electron Paramagnetic Resonance (EPR) and Circular Dichroism (CD) to assess heme environment and secondary structure.
- Enzyme activity assays to measure catalase and peroxidase function.
Main Results:
- Addition of KatG(C) to KatG(N) induced a shift in heme spin state from low-spin to high-spin, mimicking wild-type KatG.
- CD spectra indicated no significant changes in secondary structure upon domain interaction.
- Restoration of both catalase and peroxidase activities was observed in the presence of KatG(C), but not with bovine serum albumin.
- Reactivation followed slow kinetics (k(react) ≈ 4 x 10(-3) min(-1)).
Conclusions:
- The C-terminal domain is essential for restructuring the N-terminal domain into its active conformation.
- Specific interdomain interactions, rather than non-specific protein binding, are required for reconstituting the bifunctional active site.
- The process involves a slow conformational change preceding the return of catalytic activity.
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