On the contribution of the positively charged headgroup of choline to substrate binding and catalysis in the reaction catalyzed by choline oxidase.
Recent kinetic studies established that the positive charge on the trimethylammonium group of choline plays an important role in substrate binding and specificity in the reaction catalyzed by choline oxidase. In the present study, pH and solvent viscosity effects with the isosteric analogue of choline 3,3-dimethyl-butan-1-ol have been used to further dissect the contribution of the substrate positive charge to substrate binding and catalysis in the reaction catalyzed by choline oxidase. Both the kcat and kcat/Km values with 3,3-dimethyl-butan-1-ol increased to limiting values that were approximately 3- and approximately 400-times lower than those observed with choline, defining pKa values that were similar to the thermodynamic pKa value of approximately 7.5 previously determined. No effects of increased solvent viscosity were observed on the kcat and kcat/Km values with the substrate analogue at pH 8, suggesting that the chemical step of substrate oxidation is fully rate-limiting for the overall turnover and the reductive half-reaction in which the alcohol substrate is oxidized to the aldehyde. The kcat/Km value for oxygen determined with the substrate analogue was pH-independent in the pH range from 6 to 10, with an average value that was approximately 75-times lower than that previously determined with choline as substrate. These data are consistent with the positive charge headgroup of choline playing important roles for substrate binding and flavin oxidation, with minimal contribution to substrate oxidation.
Gadda
Giovanni
G
Department of Chemistry, Georgia State University, Atlanta, GA 30302-4098, USA. ggadda@gsu.edu
Fan
Fan
F
Hoang
Jane V
JV
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001173:Arthrobacter / Q000201:enzymology; D002384:Catalysis; D002412:Cations; D002794:Choline / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism*; D005415:Flavins / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D015394:Molecular Structure; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012997:Solvents / Q000737:chemistry; D013379:Substrate Specificity; D013816:Thermodynamics; D014783:Viscosity
D002412:Cations; D005415:Flavins; D011994:Recombinant Proteins; D012997:Solvents; D000429:Alcohol Oxidoreductases; C019888:choline oxidase; D002794:Choline