NDLI: Contribution of Flavin Covalent Linkage with Histidine 99 to the Reaction Catalyzed by Choline Oxidase*

Content Provider	PubMed Central
Author	Quaye, Osbourne Cowins, Sharonda Gadda, Giovanni
Abstract	The FAD-dependent choline oxidase has a flavin cofactor covalently attached to the protein via histidine 99 through an 8α-N(3)-histidyl linkage. The enzyme catalyzes the four-electron oxidation of choline to glycine betaine, forming betaine aldehyde as an enzyme-bound intermediate. The variant form of choline oxidase in which the histidine residue has been replaced with asparagine was used to investigate the contribution of the 8α-N(3)-histidyl linkage of FAD to the protein toward the reaction catalyzed by the enzyme. Decreases of 10-fold and 30-fold in the k cat/Km and k cat values were observed as compared with wild-type choline oxidase at pH 10 and 25 °C, with no significant effect on k cat/K O using choline as substrate. Both the k cat/Km and k cat values increased with increasing pH to limiting values at high pH consistent with the participation of an unprotonated group in the reductive half-reaction and the overall turnover of the enzyme. The pH independence of both D(k cat/Km ) and D k cat, with average values of 9.2 ± 3.3 and 7.4 ± 0.5, respectively, is consistent with absence of external forward and reverse commitments to catalysis, and the chemical step of CH bond cleavage being rate-limiting for both the reductive half-reaction and the overall enzyme turnover. The temperature dependence of the D k red values suggests disruption of the preorganization in the asparagine variant enzyme. Altogether, the data presented in this study are consistent with the FAD-histidyl covalent linkage being important for the optimal positioning of the hydride ion donor and acceptor in the tunneling reaction catalyzed by choline oxidase.
Related Links	http://dx.doi.org/10.1074/jbc.M109.003715
Starting Page	16990
File Format	PDF
ISSN	1083351X
e-ISSN	1083351X
Journal	The Journal of Biological Chemistry
Issue Number	25
Volume Number	284
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2009-06-19
Access Restriction	Open
Rights Holder	American Society for Biochemistry and Molecular Biology
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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Contribution of Flavin Covalent Linkage with Histidine 99 to the Reaction Catalyzed by Choline Oxidase

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