Energetics in depolymerization of recalcitrant polysaccharides

The enzymatic degradation of the closely related insoluble polysaccharides cellulose [beta(1-4) linked glucose] and chitin [beta(1-4) linked N-acetylglucosamine] is of large biological and economical importance. This process is higly demanding since enzym es acting on cellulose or chitin face the challenges of associating with the insoluble substrate, disrupting crystal packing, and guiding a single polymer chain into the catalytic centre. In addition to a high free energy penalty in decrystallization of s ingle crystalline cellulose and chitin fibrils, there is also a free energy penalty in changing the conformation of a glucose/N-acetylglucosamine moiety before hydrolysis can take place making the whole degradation process very demanding. This loss in fre e energy needs to be compensated through the free energy associated with binding of the enzyme to the substrate. Features that overcome the low accessibility of the substrate and provide necessary free energy upon binding are the presence of long and deep , sometimes tunnel-like substrate-binding site clefts lined with both aromatic amino acids that stacks with the pyranose rings and charged and polar amino acids that participates in dipolar interactions. These enzymes act processively, i.e., single carboh ydrate chains are threaded through the active site clefts, while disaccharides are cleaved off at the catalytic centre. We want to to develop simulation methodolgies on enzymatic factors in chitinase A and chitinase B of Serratia marcescens that are impo rtant for processive degradation of chitin and compare obtained theoretical data to experimental. We already have experimentally obtained quantitative data for the wild type enzymes and selected site-directed mutants and want to complement these data with data for new sets of mutants that have polar interactions with the substrate.