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On the Effects of Reversing the Protein Positive Charge in Proximity of the N(1)‐Flavin Locus in Choline Oxidase
Author(s) -
Ghanem Mahmoud,
Gadda Giovanni
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a906-d
Choline oxidase catalyzes the oxidation of choline to betaine via two sequential flavin‐linked hydride transfer reactions from choline and betaine aldehyde to oxygen ( 1, 2 ). The conserved H466 is located 3.2 Å from the N(1) locus of FAD, and was shown to be positively charged during catalysis ( 3 ). Previous data suggested that H466 modulates the electrophilicity of the flavin and the polarity of the active site, and stabilizes the choline‐alkoxide intermediate that is formed in catalysis ( 3 ). In the present study, we prepared and characterized two mutant proteins in which H466 was replaced with a neutral (CHO‐H466A) or a negatively charged (CHO‐H466D) residue. The spectral properties of the CHO‐H466D, CHO‐H466A, and WT enzymes were investigated in the oxidized, 1‐ and 2‐electron reduced forms of the flavin. The effects of pH on the absorbance spectra of the oxidized and reduced enzymes were also determined, along with the oxidation‐reduction potentials for the enzyme‐bound flavin. All taken together, the data presented further establish the importance of a positively charged residue in modulating the electrophilicity of the enzyme‐bound flavin for efficient catalysis (Scheme).