Kinetic mechanism of choline oxidase from Arthrobacter globiformis.
Choline oxidase catalyzes the four-electron oxidation of choline to glycine-betaine, with betaine-aldehyde as intermediate and molecular oxygen as primary electron acceptor. The enzyme is capable of accepting betaine-aldehyde as a substrate, allowing the investigation of the reaction mechanism for both the conversion of choline to the aldehyde intermediate and of betaine-aldehyde to glycine-betaine. The steady state kinetic mechanism has been determined at pH 7 with choline and betaine-aldehyde as substrate to be sequential, consistent with oxygen reacting with the reduced enzyme before release of betaine-aldehyde or glycine-betaine, respectively. A K(m) value < or =20 microM has been estimated for betaine-aldehyde based on the kinetic pattern with a y-intercept seen in a plot of 1/rate versus 1/[oxygen]. The kinetic data suggest that betaine-aldehyde predominantly remains bound at the active site during turnover of the enzyme with choline. In agreement with such a conclusion, less than 10% betaine-aldehyde has been found in the reaction mixture under enzymatic turnover with saturating concentrations of choline. The k(cat) values were 6.4+/-0.3 and 15.3+/-2.5 s(-1) for choline and betaine-aldehyde, respectively, suggesting that a kinetic step in the oxidation of choline to the aldehyde intermediate must be partially rate-limiting for catalysis. Cleavage of the CH bond of choline as being partially rate-limiting for catalysis is discussed.
Gadda
Giovanni
G
Department of Chemistry, Georgia State University, P.O. Box 4098, Atlanta, GA 30302-4098, USA. ggadda@gsu.edu
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D001173:Arthrobacter / Q000201:enzymology*; D001622:Betaine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D002384:Catalysis; D002794:Choline / Q000378:metabolism; D016018:Least-Squares Analysis; D008956:Models, Chemical; D010084:Oxidation-Reduction
D001622:Betaine; C026820:betaine aldehyde; D000429:Alcohol Oxidoreductases; C019888:choline oxidase; D002794:Choline