O-Nitrophenyl Cellobioside As an Active Site Probe for Family 7 Cellobiohydrolases
AIChE Annual Meeting
Monday, November 14, 2016 - 10:00am to 12:30pm
Interactions between o-nitrophenyl-Î²-D-cellobioside (oNPC) and two cellobiohydrolases, Cel7A from Hypocrea jecorina (Trichoderma reesei) and Cel7D from Phanerochaete chrysosporium were studied by fluorescence spectroscopy and kinetic tests. The hydrolysis rate is slow enough that the substance may be treated as nonreactive in equilibrium binding studies. oNPC quenches the natural fluorescence of Cel7A and Cel7D by radiationless energy transfer. Addition of cellobiose recovers the fluorescence of Cel7A, indicating that oNPC binds preferentially to the +1,+2 subsites of the enzyme, to which cellobiose is known to have highest affinity. oNPC was used as indicator ligand to determine the association constants of cellobiose for catalytically inactive mutants of Cel7A by displacement binding experiment. The experimental data are supplemented by structural modelling of oNPC into the active site of the enzymes studied. Further, molecular dynamics simulations were performed to identify active site contributions to affinity, and free energy calculations (molecular mechanics Poisson-Boltzmann surface area) were conducted to understand preferential subsite binding. Kinetic studies have shown that oNPC is far less susceptible to hydrolysis by Cel7A than other active site probes. Molecular dynamics simulation has shown a wealth of hydrogen bonds formed and highly favorable interaction energy when oNPC is bound in the +1,+2 subsites.