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On the Catalytic Role of the Conserved Active Site Residue His466 of Choline Oxidase.
- Source :
-
Biochemistry . 1/25/2005, Vol. 44 Issue 3, p893-904. 12p. - Publication Year :
- 2005
-
Abstract
- The oxidation of alcohols to aldehydes is catalyzed by a number of flavin-dependent enzymes, which have been grouped in the glucose-methanolch oxidoreductase enzyme superfamily. These enzymes exhibit little sequence similarity in their substrates binding domains, but share a highly conserved catalytic site, suggesting a similar activation mechanism for the oxidation of their substrates. In this study, the fully conserved histidine residue at position 466 of choline oxidase was replaced with an alanine residue by site-directed mutagenesis and the biochemical, spectroscopic, and mechanistic properties of the resulting CHO-H466A mutant enzyme were characterized, CHO-H466A showed kcat and kcat/Km values with choline as substrate that were 60- and 1000-fold lower than the values for the wild-type enzyme, while the kcajKm value for oxygen was unaffected, suggesting the involvement of His466 in the oxidation of the alcohol substrate but not in the reduction of oxygen. Replacement of His4 with alanine significantly affected the microenvironment of the flavin, as indicated by the altered behavior of CHO-H466A with sulfite and dithionite. In agreement with this conclusion, a midpoint reduction potential of +106 mV for the two-electron transfer in the catalytically competent enzyme-product complex was determined at pH 7 for CHO-H466A, which was `-`-`25 mV more negative than that of the wild-type enzyme. Enzymatic activity in CHO-H466A could be partially rescued with exogenous imidazolium, but not imidazole, consistent with the protonated form of histidine exerting a catalytic role. pH profiles for glycine betaine inhibition, the deprotonation of the N(3)-flavin locus, and the kcatlKm value for choline all showed a significant shift upward in their pK0 values, consistent with a change in the polarity of the active site. Finally, kinetic isotope effects with isotopically labeled substrate and solvent indicated that he histidine to alanine substitution affected the timing of substrate OH and CH bond cleavages, consistent with removal of the hydroxyl proton being concerted with hydride transfer in the mutant enzyme. All taken together, the results presented in this study suggest that in choline oxidase, His466 modulates the electrophilicity of the enzyme-bound flavin and the polarity of the active site, and contributes to the stabilization of the transition state for the oxidation of choline to betaine aldehyde. [ABSTRACT FROM AUTHOR]
- Subjects :
- *BINDING sites
*ENZYMES
*OXIDOREDUCTASES
*BIOCHEMISTRY
*FLAVINS
*COENZYMES
Subjects
Details
- Language :
- English
- ISSN :
- 00062960
- Volume :
- 44
- Issue :
- 3
- Database :
- Academic Search Index
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 16049117
- Full Text :
- https://doi.org/10.1021/bi048056j