Involvement of ionizable groups in catalysis of human liver glycolate oxidase.
Glycolate oxidase is a flavin-dependent, peroxisomal enzyme that oxidizes alpha-hydroxy acids to the corresponding alpha-keto acids, with reduction of oxygen to H(2)O(2). In plants, the enzyme participates in photorespiration. In humans, it is a potential drug target for treatment of primary hyperoxaluria, a genetic disorder where overproduction of oxalate results in the formation of kidney stones. In this study, steady-state and pre-steady-state kinetic approaches have been used to determine how pH affects the kinetic steps of the catalytic mechanism of human glycolate oxidase. The enzyme showed a Ping-Pong Bi-Bi kinetic mechanism between pH 6.0 and 10.0. Both the overall turnover of the enzyme (k(cat)) and the rate constant for anaerobic substrate reduction of the flavin were pH-independent at pH values above 7.0 and decreased slightly at lower pH, suggesting the involvement of an unprotonated group acting as a base in the chemical step of glycolate oxidation. The second-order rate constant for capture of glycolate (k(cat)/K(glycolate)) and the K(d)((app)) for the formation of the enzyme-substrate complex suggested the presence of a protonated group with apparent pK(a) of 8.5 participating in substrate binding. The k(cat)/K(oxygen) values were an order of magnitude faster when a group with pK(a) of 6.8 was unprotonated. These results are discussed in the context of the available three-dimensional structure of GOX.
Pennati
Andrea
A
Department of Chemistry, Georgia State University, Atlanta, Georgia 30302-4098, USA.
Gadda
Giovanni
G
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D004795:Enzyme Stability; D005415:Flavins / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000737:chemistry / Q000201:enzymology*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D005415:Flavins; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C020821:glycollate oxidase