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Spectroscopic and kinetic properties of recombinant choline oxidase from Arthrobacter globiformis.
Biochemistry. 2003 Dec 30; 42(51):15179-88.B

Abstract

Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, with molecular oxygen acting as primary electron acceptor. Recently, the recombinant enzyme expressed in Escherichia coli was purified to homogeneity and shown to contain FAD in a mixture of oxidized and anionic semiquinone redox states [Fan et al. (2003) Arch. Biochem. Biophys., in press]. In this study, methods have been devised to convert the enzyme-bound flavin semiquinone to oxidized FAD and vice versa, allowing characterization of the resulting forms of choline oxidase. The enzyme-bound oxidized flavin showed typical UV-vis absorbance peaks at 359 and 452 nm (with epsilon(452) = 11.4 M(-1) cm(-1)) and emitted light at 530 nm (with lambda(ex) at 452 nm). The affinity of the enzyme for sulfite was high (with a K(d) value of approximately 50 microM at pH 7 and 15 degrees C), suggesting the presence of a positive charge near the N(1)C(2)=O locus of the flavin. The enzyme-bound anionic flavin semiquinone was unusually insensitive to oxygen or ferricyanide at pH 8 and showed absorbance peaks at 372 and 495 nm (with epsilon(372) = 19.95 M(-1) cm(-1)), maximal fluorescence emission at 454 nm (with lambda(ex) at 372 nm), circular dichroic signals at 370 and 406 nm, and an ESR peak-to-peak line width of 13.9 G. Both UV-vis absorbance studies on the enzyme under turnover with choline and steady-state kinetic data with either choline or betaine aldehyde were consistent with the flavin semiquinone being not involved in catalysis. The pH dependence of the kinetic parameters at varying concentrations of both choline and oxygen indicated that a catalytic base is required for choline oxidation but not for oxygen reduction and that the order of the kinetic steps involving substrate binding and product release is not affected by pH.

Authors+Show Affiliations

Department of Chemistry, The Center for Biotechnology and Drug Design, Georgia State University, Atlanta, Georgia 30302-4098, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

14690428

Citation

Ghanem, Mahmoud, et al. "Spectroscopic and Kinetic Properties of Recombinant Choline Oxidase From Arthrobacter Globiformis." Biochemistry, vol. 42, no. 51, 2003, pp. 15179-88.
Ghanem M, Fan F, Francis K, et al. Spectroscopic and kinetic properties of recombinant choline oxidase from Arthrobacter globiformis. Biochemistry. 2003;42(51):15179-88.
Ghanem, M., Fan, F., Francis, K., & Gadda, G. (2003). Spectroscopic and kinetic properties of recombinant choline oxidase from Arthrobacter globiformis. Biochemistry, 42(51), 15179-88.
Ghanem M, et al. Spectroscopic and Kinetic Properties of Recombinant Choline Oxidase From Arthrobacter Globiformis. Biochemistry. 2003 Dec 30;42(51):15179-88. PubMed PMID: 14690428.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Spectroscopic and kinetic properties of recombinant choline oxidase from Arthrobacter globiformis. AU - Ghanem,Mahmoud, AU - Fan,Fan, AU - Francis,Kevin, AU - Gadda,Giovanni, PY - 2003/12/24/pubmed PY - 2004/5/5/medline PY - 2003/12/24/entrez SP - 15179 EP - 88 JF - Biochemistry JO - Biochemistry VL - 42 IS - 51 N2 - Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, with molecular oxygen acting as primary electron acceptor. Recently, the recombinant enzyme expressed in Escherichia coli was purified to homogeneity and shown to contain FAD in a mixture of oxidized and anionic semiquinone redox states [Fan et al. (2003) Arch. Biochem. Biophys., in press]. In this study, methods have been devised to convert the enzyme-bound flavin semiquinone to oxidized FAD and vice versa, allowing characterization of the resulting forms of choline oxidase. The enzyme-bound oxidized flavin showed typical UV-vis absorbance peaks at 359 and 452 nm (with epsilon(452) = 11.4 M(-1) cm(-1)) and emitted light at 530 nm (with lambda(ex) at 452 nm). The affinity of the enzyme for sulfite was high (with a K(d) value of approximately 50 microM at pH 7 and 15 degrees C), suggesting the presence of a positive charge near the N(1)C(2)=O locus of the flavin. The enzyme-bound anionic flavin semiquinone was unusually insensitive to oxygen or ferricyanide at pH 8 and showed absorbance peaks at 372 and 495 nm (with epsilon(372) = 19.95 M(-1) cm(-1)), maximal fluorescence emission at 454 nm (with lambda(ex) at 372 nm), circular dichroic signals at 370 and 406 nm, and an ESR peak-to-peak line width of 13.9 G. Both UV-vis absorbance studies on the enzyme under turnover with choline and steady-state kinetic data with either choline or betaine aldehyde were consistent with the flavin semiquinone being not involved in catalysis. The pH dependence of the kinetic parameters at varying concentrations of both choline and oxygen indicated that a catalytic base is required for choline oxidation but not for oxygen reduction and that the order of the kinetic steps involving substrate binding and product release is not affected by pH. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/14690428/Spectroscopic_and_kinetic_properties_of_recombinant_choline_oxidase_from_Arthrobacter_globiformis_ L2 - https://doi.org/10.1021/bi035435o DB - PRIME DP - Unbound Medicine ER -