Preparation and characterization of cobalt-substituted anthrax lethal factor
Crystal E Säbel1, Ryan Carbone, John R Dabous
1Department of Chemistry and Biochemistry, Laurentian University, 935 Ramsey Lake Rd., Sudbury, Ontario, Canada P3E 2C6.
Biochemical and Biophysical Research Communications
|November 19, 2011
Summary
Cobalt-substituted anthrax lethal factor (CoLF) was created and found to be twice as active as native zinc-LF. This CoLF serves as the first spectroscopic probe of the enzyme's active site.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Anthrax lethal factor (LF) is a zinc-dependent endopeptidase.
- LF cleaves mitogen-activated protein kinase kinases, impacting cellular signaling.
Purpose of the Study:
- To prepare and characterize cobalt-substituted LF (CoLF).
- To investigate the electronic properties and activity of CoLF.
- To explore the mechanism of metal removal from LF.
Main Methods:
- Preparation of CoLF via bio-assimilation or direct metal exchange.
- Characterization using electronic spectroscopy.
- Assessment of enzymatic activity compared to zinc-LF.
- Spectroscopic studies with thioglycolic acid (TGA) to probe metal removal.
Main Results:
- CoLF was successfully prepared using two distinct methods.
- Each CoLF molecule contains one Co(2+) ion.
- CoLF exhibited twice the enzymatic activity of native zinc-LF.
- Electronic spectroscopy indicated a five-coordinate Co(2+) ion in CoLF.
- A sequential mechanism for metal removal by TGA was elucidated.
Conclusions:
- CoLF is a more active and spectroscopically informative variant of LF.
- The Co(2+) coordination in CoLF differs from native LF's crystal structure.
- Spectroscopic analysis of CoLF provides a novel tool for studying LF's active site dynamics and inhibitor interactions.
