Abstract
Owing to their ability to break glycosidic bonds in recalcitrant crystalline polysaccharides such as cellulose, the catalysis effected by lytic polysaccharide monooxygenases (LPMOs) is of major interest. Kinetics of these reductant-dependent, monocopper enzymes is complicated by the insoluble nature of the cellulose substrate and parallel, enzyme-dependent, and enzyme-independent side reactions between the reductant and oxygen-containing cosubstrates. Here, we provide kinetic characterization of cellulose peroxygenase (oxidative cleavage of glycosidic bonds in cellulose) and reductant peroxidase (oxidation of the reductant) activities of the LPMO TrAA9A of the cellulose-degrading model fungus Trichoderma reesei. The catalytic efficiency of the cellulose peroxygenase reaction (kcat = 8.5 s−1, and ) was an order of magnitude higher than that of the reductant (ascorbic acid) peroxidase reaction. The turnover of H2O2 in the ascorbic acid peroxidase reaction followed the ping-pong mechanism and led to irreversible inactivation of the enzyme with a probability of 0.0072. Using theoretical analysis, we suggest a relationship between the half-life of LPMO, the values of kinetic parameters, and the concentrations of the reactants.
Highlights
Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that catalyze oxidative cleavage of glycosidic bonds in various polysaccharides [1,2,3,4,5]
The kcat =KmðH2O2Þ value of 0.29 μM−1 s−1 measured on bacterial microcrystalline cellulose (BMCC) (Table 1) is well in line with the kcat =KmðH2O2Þ value of 0.27 μM−1 s−1 measured for the same enzyme on wood-derived cellulose (Avicel) using competition experiments with other H2O2-consuming enzymes [22]
Because the reductant peroxidase reaction can lead to the irreversible inactivation of LPMO, an in-depth understanding of the kinetics and mechanism of this reaction is of utmost importance regarding the application of LPMOs
Summary
Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that catalyze oxidative cleavage of glycosidic bonds in various polysaccharides [1,2,3,4,5]. The apparent kcat =KmðH2O2Þ of the cellulolytic peroxygenase reaction is independent on the kinetic parameters of the reduction and reoxidation of the active site copper as well as on the concentration of the reductant (Fig. 5B).
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