title: Kinetic mechanism of L-α-glycerophosphate oxidase from Mycoplasma pneumoniae.
abstract: L-α-glycerophosphate oxidase is an FAD-dependent enzyme that catalyzes the oxidation of L-α-glycerophosphate (Glp) by molecular oxygen to generate dihydroxyacetone phosphate (DHAP) and hydrogen peroxide (H2O2). The catalytic properties of recombinant His6-GlpO from Mycoplasma pneumoniae (His6-MpGlpO) were investigated through transient and steady-state kinetics and ligand binding studies. The results indicate that the reaction mechanism of His6-MpGlpO follows a ping-pong model. Double-mixing mode stopped-flow experiments show that, after flavin-mediated substrate oxidation, DHAP leaves rapidly prior to the oxygen reaction. The values determined for the individual rate constants and kcat (4.2 s(-1) at 4 °C), in addition to the finding that H2 O2 binds to the oxidized enzyme, suggest that H2O2 release is the rate-limiting step for the overall reaction. The results indicate that His6 -MpGlpO contains mixed populations of fast- and slow-reacting species. It is predominantly the fast-reacting species that participates in turnover. In contrast to other GlpO enzymes previously described, His6-MpGlpO is able to catalyze the reverse reaction of reduced enzyme and DHAP. This result may be explained by the standard reduction potential value of His6-MpGlpO (-167 ± 1 mV), which is lower than those of GlpO from other species. We found that D,L-glyceraldehyde 3-phosphate (GAP) may be used as a substrate in the His6-MpGlpO reaction, although it exhibited an approximately 100-fold lower kcat value in comparison with the reaction of Glp. These results also imply involvement of GlpO in glycolysis, as well as in lipid and glycerol metabolism. The kinetic models and distinctive properties of His6-MpGlpO reported here should be useful for future drug development against Mycoplasma pneumoniae infection.
authors.affiliation: Department of Biochemistry, Faculty of Science, Burapha University, Chonburi, Thailand.
authors.forename: Somchart
authors.initials: S
authors.lastname: Maenpuen
authors.affiliation: Department of Biochemistry, Faculty of Science, Burapha University, Chonburi, Thailand.
authors.forename: Pratchaya
authors.initials: P
authors.lastname: Watthaisong
authors.affiliation: Department of Biochemistry, Faculty of Science, Burapha University, Chonburi, Thailand.
authors.forename: Pacharee
authors.initials: P
authors.lastname: Supon
authors.affiliation: Department of Biochemistry, Faculty of Dentistry, Chulalongkorn University, Patumwan, Bangkok, Thailand.
authors.forename: Jeerus
authors.initials: J
authors.lastname: Sucharitakul
authors.affiliation: Department of Biochemistry and Center for Structural Biology, Wake Forest School of Medicine, Winston-Salem, NC, USA.
authors.forename: Derek
authors.initials: D
authors.lastname: Parsonage
authors.affiliation: Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA.
authors.forename: P Andrew
authors.initials: PA
authors.lastname: Karplus
authors.affiliation: Department of Biochemistry and Center for Structural Biology, Wake Forest School of Medicine, Winston-Salem, NC, USA.
authors.forename: Al
authors.initials: A
authors.lastname: Claiborne
authors.affiliation: Department of Biochemistry and Center of Excellence in Protein Structure & Function, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors.forename: Pimchai
authors.initials: P
authors.lastname: Chaiyen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004099:Dihydroxyacetone Phosphate / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005986:Glyceraldehyde 3-Phosphate / Q000378:metabolism; D005993:Glycerolphosphate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D009177:Mycoplasma pneumoniae / Q000201:enzymology* / Q000235:genetics; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D011994:Recombinant Proteins; D005986:Glyceraldehyde 3-Phosphate; D005182:Flavin-Adenine Dinucleotide; D004099:Dihydroxyacetone Phosphate; D006861:Hydrogen Peroxide; D005993:Glycerolphosphate Dehydrogenase; C098831:glycerol-3-phosphate oxidase; D010100:Oxygen
keywords: Mycoplasma pneumoniae; flavin adenine dinucleotide (FAD); flavoprotein oxidase; l-α-glycerophosphate oxidase; transient kinetics