LAUSR

Polysaccharide monooxygenases (PMOs) are mononuclear copper enzymes that catalyze the hydroxylation of polysaccharides leading to the scission of the glycosidic bond. The mechanism, in which PMOs utilize molecular oxygen to oxidize the polysaccharide substrate, still remains largely unknown. Here, steady-state kinetics assays were used to probe the mechanism of oxygen-dependent cellohexaose oxidation catalyzed by MtPMO9E. Kinetic analysis indicated that both kcat/ KM(O2) and kcat/ KM(Glc6) were dependent on the concentration of the second substrate. Inhibition studies using carbon monoxide were also carried out. In addition, KD values for Glc6 were determined for the Cu(I) and Cu(II) forms of the enzyme. Taken together, PMOs follow a random-sequential kinetic mechanism to form a ternary ES-O2 complex. The optimal pH for MtPMO9E turnover was determined to be between pH 6.00 and pH 7.00. Furthermore, the kinetic parameters kcat, kcat/ KM(O2), and kcat/ KM(Glc6) demonstrate a decrease in PMO activity at a low pH and provide equivalent kinetic p Ka's of 5.10. This points to the protonation of a general base required for turnover. These results provide a basis for the initial chemical steps in the mechanism of PMOs.