Crystalline Pyruvate Oxidase from Escherichia coli
Year: 1971
Type: article
Abstract: Crystalline pyruvate oxidase, a soluble tetrameric flavoprotein from Escherichia coli, which binds both thiamine pyrophosphate (TPP) and FAD is activated 15- to 100-fold by phospholipids and long chain fatty acids. Maximal activation of the oxidase requires incubation of the enzyme for at least 6 min with the lipid activator in the presence of substrate and cofactors (pyruvate, TPP, and MgCl2). Very little activation occurs if any of these components are omitted from the mixture. Furthermore, activation is markedly reduced if the enzyme is incubated with phospholipid before pyruvate, TPP, and MgCl2 are added. Phosphatides dramatically effect the kinetic parameters of the pyruvate oxidase reaction. The Km values for pyruvate and TPP are lowered 13-fold and 3- to 4-fold, respectively, in the presence of phospholipid. In addition, phosphatides bestow cooperativity to the enzyme with respect to binding of TPP in a very unusual fashion. In the absence of phospholipid, TPP binding to the enzyme follows ordinary Michaelis-Menten type saturation kinetics. In the presence of phospholipid, TPP binds cooperatively to the enzyme and shifts the Km for TPP to a lower value. Stopped-flow experiments measuring the rate of reduction of enzyme-bound FAD clearly indicate that the presence of lipid activator affects a rate-controlling step leading to the formation of enzyme-FADH2. The rate of reduction of enzyme-bound FAD is increased at least 100-fold in the presence of phospholipid. The physiological significance of pyruvate oxidase and the activation phenomenon are discussed.
Source: Journal of Biological Chemistry
Authors: C.C. Cunningham, Lowell P. Hager
Institution: University of Illinois Urbana-Champaign
Language: English
Subfield: Biochemistry
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W1522246139