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|Title:||Circular dichroism studies of Escherichia coli pyruvate oxidase and secondary structure and lipid binding properties of the alpha peptide from pyruvate oxidase|
|Author(s):||Schuh, William Kenneth|
|Doctoral Committee Chair(s):||Hager, Lowell P.|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Pyruvate oxidase, a tetrameric enzyme consisting of four identicle subunits, undergoes a change in conformation with the binding of TPP and pyruvate. Circular dichroism measurements show the change in secondary structure with this ligand induced conformational change. Circular dichroism spectra also change with the addition of phospholipid vesicles which bring about an increase in catalytic activity. The circular dichroism spectra for detergent activated and proteolytically activated pyruvate oxidase are virtually identicle indicating there is a further change to the maximally activated conformation of the enzyme. Circular dichroism spectra obtained with varying levels of phospholipid vesicles which activate the enzyme to varying levels give patterns which vary between the minimally and maximally activated spectra. These spectra positively correlate with the level of enzyme activity. Activation of pyruvate oxidase with HFP, a hydrophobic region disruptor, also alters the circular dichroism spectra to a degree consistent with that of lipid activation at similar activity levels. This indicates that the removal of the alpha-peptide region of pyruvate oxidase from the catalytic site brings about a change in conformation which maximally activates the enzyme. Lower activity levels is seen with smaller changes in conformation.
Proteolytic activation of pyruvate oxidase with $\alpha$-chymotrypsin in the presence of TPP and pyruvate releases a 23 residue peptide, the alpha-peptide, from the carboxyl terminus. This peptide was purified in its entirety. Circular dichroism studies of this peptide reveal that it is primarily a random chain with $\alpha$-helix present at 18.1%. This amphipathic helix increases with the binding of phospholipid vesicles to 25.2%.
Fluorescence energy transfer studies using DDA at the surface and 16-AP at the midplane of the phospholipid vesicles determined that the single tryptophan residue located at the third position from the carboxyl terminus lies at a depth of 10.5 A from the surface.
The alpha-peptide binds stably to the phospholipid vesicles, yet the removal of the 2 carboxyl terminal residues decreases binding affinity by approximately 75%. A synthetic peptide with a proline inserted into the center of the amphipathic helix decreased helicity and lipid binding by roughly 30%. A peptide with a glycine replacing the carboxyl terminal arginine did not change the helical content, yet the lipid binding was decreased by 50%. This indicates that the amphipathic helix, the carboxyl terminal positive charge, and carboxyl terminal hydrophobic residues all play a role in the lipid binding properties of the alpha-peptide.
|Rights Information:||Copyright 1992 Schuh, William Kenneth|
|Date Available in IDEALS:||2011-05-07|
|Identifier in Online Catalog:||AAI9305685|