Contribution of flavin covalent linkage with histidine 99 to the reaction catalyzed by choline oxidase.
The FAD-dependent choline oxidase has a flavin cofactor covalently attached to the protein via histidine 99 through an 8alpha-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 8alpha-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)/K(m) and k(cat) values were observed as compared with wild-type choline oxidase at pH 10 and 25 degrees C, with no significant effect on k(cat)/K(O) using choline as substrate. Both the k(cat)/K(m) 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)/K(m)) 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.
Quaye
Osbourne
O
From the Departments of Chemistry, Atlanta, Georgia 30302-4098.
Cowins
Sharonda
S
From the Departments of Chemistry, Atlanta, Georgia 30302-4098; Department of Chemistry, Albany State University, Albany, Georgia 31705.
Gadda
Giovanni
G
From the Departments of Chemistry, Atlanta, Georgia 30302-4098; Biology, Atlanta, Georgia 30302-4098; The Center for Biotechnology and Drug Design, Georgia State University, Atlanta, Georgia 30302-4098. Electronic address: ggadda@gsu.edu.
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D001173:Arthrobacter / Q000201:enzymology / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D006639:Histidine; D000429:Alcohol Oxidoreductases; C019888:choline oxidase