Pyruvate formate-lyase-activating enzyme: strictly anaerobic isolation yields active enzyme containing a [3Fe-4S](+)

¹Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA. broderj@cem.msu.edu

Biochemical and Biophysical Research Communications

|March 10, 2000

Summary

Pyruvate formate-lyase-activating enzyme (PFL-AE) was successfully isolated under anaerobic conditions, revealing its essential [3Fe-4S](+) cluster. This native cluster is crucial and sufficient for the enzyme's catalytic activity.

Area of Science:

Biochemistry
Enzymology
Protein Chemistry

Background:

Pyruvate formate-lyase-activating enzyme (PFL-AE) from Escherichia coli (E. coli) is critical for pyruvate formate-lyase (PFL) activation.
Enzyme activity is dependent on S-adenosyl-l-methionine (AdoMet) and an iron-dependent cofactor.
Previous challenges included isolating PFL-AE with its labile metal center intact due to oxygen sensitivity.

Purpose of the Study:

To report the first successful isolation of PFL-AE under nondenaturing, anaerobic conditions.
To characterize the metal center present in the purified enzyme.
To determine the necessity and sufficiency of the native metal cluster for enzymatic activity.

Main Methods:

Strictly anaerobic and nondenaturing purification techniques.
Iron and sulfide content analysis.
Spectroscopic characterization including UV-visible, EPR, and resonance Raman spectroscopy.

Main Results:

PFL-AE was isolated with its metal center intact under anaerobic conditions.
Analysis confirmed the presence of a [3Fe-4S](+) cluster in the purified enzyme.
The isolated native enzyme demonstrated high specific activity (31 U/mg) without added iron, unlike apo-enzyme.

Conclusions:

The native [3Fe-4S](+) cluster is essential and sufficient for PFL-AE activity.
This study provides a method for isolating active PFL-AE, overcoming previous limitations.
Understanding the cofactor is key to elucidating PFL-AE's catalytic mechanism.

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Pyruvate formate-lyase-activating enzyme: strictly anaerobic isolation yields active enzyme containing a [3Fe-4S](+)...

Area of Science:

Background:

Purpose of the Study:

Main Methods:

Main Results:

Conclusions:

Related Concept Videos

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Pyruvate formate-lyase-activating enzyme: strictly anaerobic isolation yields active enzyme containing a [3Fe-4S](+)