On the contribution of the positively charged headgroup of choline to substrate binding and catalysis in the reaction catalyzed by choline oxidase q Giovanni Gadda a,b,c, * , Fan Fan b,1 , Jane V. Hoang a,1 a Department of Chemistry, Georgia State University, Atlanta, GA 30302-4098, USA b Department of Biology, Georgia State University, Atlanta, GA 30302-4098, USA c Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30302-4098, USA Received 1 March 2006, and in revised form 7 April 2006 Available online 3 May 2006 Abstract 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 char- ge to substrate binding and catalysis in the reaction catalyzed by choline oxidase. Both the k cat and k cat /K m values with 3,3-dimethyl- butan-1-ol increased to limiting values that were 3- and 400-times lower than those observed with choline, defining pK a values that were similar to the thermodynamic pK a value of 7.5 previously determined. No effects of increased solvent viscosity were observed on the k cat and k cat /K m 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 k cat / K m 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 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. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Choline oxidase; Solvent viscosity; Substrate binding; Catalysis; Flavoprotein; Substrate analogue Choline oxidase (E.C. 1.1.3.17) catalyzes the two-step oxidation of choline to glycine betaine (N,N,N-trimethyl- glycine; betaine) via betaine aldehyde as intermediate (Scheme 1) [1]. The enzymatic turnover is mediated by FAD and utilizes molecular oxygen as primary electron acceptor [2–4]. The enzyme is of both biotechnological and biomedical interests because the reaction product, gly- cine betaine, is one in a limited number of compounds that accumulate to high levels in the cytoplasm of cells to pre- vent dehydration and plasmolysis in adverse hyperosmotic environments [5–8] in pathogenic bacteria [9–11] and trans- genic plants [12–19]. Consequently, the study of choline oxidase has potential for the development of therapeutic agents that inhibit glycine betaine biosynthesis and render pathogenic bacteria susceptible to either conventional treatments or the immune system, and for the engineering of drought and temperature resistance in economically rel- evant crops. Choline oxidase from Arthrobacter globiformis strain ATCC 8010 has been previously cloned and expressed to www.elsevier.com/locate/yabbi Archives of Biochemistry and Biophysics 451 (2006) 182–187 ABB 0003-9861/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2006.04.005 q This work was supported in part by Grants PRF #37351-G4 and PRF #43763-AC4 from the American Chemical Society, and a Research Initiation Grant from Georgia State University (to G.G.), by a Molecular Basis of Diseases Fellowship from Georgia State University (to F.F.), and by a McNair Research Fellowship (to J.V.H.). * Corresponding author. Fax: +1 404 651 2751. E-mail address: ggadda@gsu.edu (G. Gadda). 1 These authors contributed equally to this study.