PROGRESS-CURVE ANALYSIS SHOWS THAT GLUCOSE INHIBITS THE CELLOTRIOSE HYDROLYSIS CATALYZED BY CELLOBIOHYDROLASE-II FROM TRICHODERMA-REESEI

NMR spectroscopy and HPLC were used to investigate the hydrolysis of cellotriose by cellobiohydrolase II from Trichoderma reesei. Substrate and product concentrations were followed as a function of time. Progress curves were calculated by forward numerical integration of the full kinetic equations and were fitted to the experimental data. Binding and rate constants were obtained from this fit, whereby no initial slope or Michaelis-Menten approximation was used. The progress curves from a single experiment sufficed to produce agreement with the Michaelis-Menten model (eight experiments). The absence of a kinetic isotope effect was proven. The progress-curve analysis showed that a simple degradation model cannot describe the experimental time-courses at substrate concentrations greater than 1 mM. A model containing competitive inhibition from cellobiose as well as non-competitive inhibition from glucose was developed. This four-parameter model accurately reproduces about 1000 experimental data points covering five orders of magnitude in oligosaccharide concentrations. Glucose binding to the enzyme/cellotriose complex retards, in a noncompetitive fashion, cellotriose hydrolysis by at least a factor of 30. A structural model for the noncompetitive inhibition is discussed. The NMR experiment also produced individual progress curves for the alpha and beta anomers. The beta anomer of cellotriose was degraded 2.5-times faster than the alpha anomer.