Kinetic mechanism of l -α-glycerophosphate oxidase from Mycoplasma pneumoniae

Abstract

© 2015 FEBS. 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 (H<inf>2</inf>O<inf>2</inf>). The catalytic properties of recombinant His<inf>6</inf>-GlpO from Mycoplasma pneumoniae (His<inf>6</inf>-MpGlpO) were investigated through transient and steady-state kinetics and ligand binding studies. The results indicate that the reaction mechanism of His<inf>6</inf>-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 k<inf>cat</inf> (4.2 s^-1 at 4 C), in addition to the finding that H<inf>2</inf>O<inf>2</inf> binds to the oxidized enzyme, suggest that H<inf>2</inf>O<inf>2</inf> release is the rate-limiting step for the overall reaction. The results indicate that His<inf>6</inf>-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, His<inf>6</inf>-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 His<inf>6</inf>-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 His<inf>6</inf>-MpGlpO reaction, although it exhibited an approximately 100-fold lower k<inf>cat</inf> 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 His<inf>6</inf>-MpGlpO reported here should be useful for future drug development against Mycoplasma pneumoniae infection.