Identification of a catalytic base for sugar oxidation in the pyranose 2-oxidase reaction.
Pyranose 2-oxidase (P2O) catalyzes the oxidation of aldopyranoses to form 2-keto sugars and H(2)O(2) . In this study, the mechanistic role of the conserved residues His548 and Asn593 in P2O was investigated by using site-directed mutagenesis, transient kinetics, and pH-dependence studies. As single mutants of H548 resulted in mixed populations of noncovalently bound and covalently linked FAD, double mutants containing H167A were constructed, in which the covalent histidyl-FAD linkage was removed in addition to having the H548 mutation. Single mutants H548A, H548N, H548S, H548D and double mutants (with H167A) could not be reduced by D-glucose. For the H167A/H548R mutant, the flavin could be reduced by D-glucose with the reduction rate constant about 220 times lower than that of the H167A mutant. The pH-dependence studies of H167A/H548R indicated that the rate constant of flavin reduction increased about 360-fold upon a pH rise corresponding to pK(a) >10.1, whereas the reactions of the wild-type and H167A mutant enzymes were pH independent. Therefore, the data suggest that a pK(a) value of >10.1 in the mutant enzyme is associated with the Arg548 residue, and that this residue must be unprotonated to efficiently catalyze flavin reduction. The data imply that for the wild-type P2O, the conserved His548 should be unprotonated in the pH range studied. The unprotonated His548 can act as a general base to abstract the 2-hydroxyl proton of D-glucose and initiate hydride transfer from the substrate to the flavin. Studies of the single mutant N593H showed that the flavin reduction rate constant was 114 times lower than that of the wild-type enzyme and was pH independent, while the K(d) for D-glucose binding was 19 times greater.
Department of Biochemistry and Center of Excellence in Protein Structure and Function, Mahidol University, Bangkok 10400, Thailand.
Thanyaporn
T
Wongnate
Jeerus
J
Sucharitakul
Pimchai
P
Chaiyen
D019943:Amino Acid Substitution; D055162:Biocatalysis; D002237:Carbohydrate Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D002241:Carbohydrates / Q000737:chemistry*; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D002241:Carbohydrates; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase; D005947:Glucose