Pyruvate oxidase is a peripheral membrane enzyme that can be purified to homogeneity in the absence of detergents. Once removed from the membrane particulate fraction of the cell, the activity of the oxidase in a dye-mediated assay is stimulated 20- to 50-fold by incubation with a wide variety of amphiphiles. Over 120 amphiphilic compounds have been screened for their ability to activate the oxidase, and approximately two-thirds of them were activators. The ability of amphiphiles to activate the enzyme is independent of their charge or physical state. The effect of hydrocarbon chain length upon activation has been studied using homologous series of alkyl sulfates, sulfonates, carboxylic acids, trimethylammonium bromides, and lysophosphatidylcholines, all of which activate the enzyme as monomers. A plot of the log of the concentration at which half-maximal activation is obtained versus the chain length of the activator yields a straight line, indicating an average binding free energy of -700 cal/mol of methylene group. These data are interpreted to mean that the activator binding sites on the protein are hydrophobic in nature and that the polar head group makes an unfavorable contribution to the binding. Activation studies with amphiphiles in an aggregated state, either micelles or phospholipid bilayer vesicles, indicate that the enzyme interacts preferentially with negatively charged surfaces but shows no response to differences in the fluidity of the interior of the aggregate. Activation of the enzyme with monomeric amphiphiles leads to an activated enzyme exhibiting Michaelis-Menten kinetics with respect to both pyruvate and thiamin pyrophosphate. Activation by aggregated amphiphiles yields a kinetic form of the enzyme characterized by Michaelis-Menten kinetics with respect to pyruvate and cooperative behavior with respect to thiamin pyrophosphate.
|Original language||English (US)|
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|State||Published - 1978|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology