Biophysical Characterization of an Bifunctional Iron Regulating Enzyme
Abstract
In recent years, proteins have been discovered that can interchange forms and conduct two distinct functions, One such polypeptide with two specific functions is the mammalian iron responsive element (IRE) binding protein IRP-1. When iron levels within the cell are normal, this protein contains an iron-sulfur cluster and is known as cytoplasmic aconitase, an enzyme that catalyzes a specific chemical reaction. When iron levels fall below normal, however, the iron-sulfur cluster is lost, the protein loses its enzymatic activity and it becomes IRP-1, which binds to certain regions of ribonucleic acid (RNA). This binding action helps restore iron levels to normal, and in the process returns the protein to its cytoplasmic aconitase form. In order to understand the structural features that stabilize this large bifunctional protein, folding studies were performed in the presence and absence of iron on a recombinant version of the human protein. Equilibrium unfolding experiments in urea revealed that human cytoplasmic aconitase undergoes a multi-state transition from the native to denatured form. Comparison of unfolding in the presence and absence of the 4Fe-4S cluster indicate that the cluster does not have a significant effect on the equilibrium unfolding properties as monitored by fluorescence. Monitoring enzymatic activity as a function of urea concentration indicates that activity is lost at ^2M urea, presumably due to disruption of the enzyme active site, which is at the major interdomain cleft. The effect of thermal denaturation on the stability and activity of the IRP-1 was also studied. A preliminary model for the unfolding of aconitase based on its enzyme activity, equilibrium thermal properties, and chemical denaturation data is presented.
Document Details
- Document Type
- Technical Report
- Publication Date
- May 01, 2002
- Accession Number
- ADA406568
Entities
People
- Pritha M. Mahadevan
Organizations
- United States Naval Academy