P65: Determination of thermodynamic and kinetic parameters of polygalacturonase enzymes acting on galacturonic acid oligomers using isothermal titration calorimetry

Polygalacturonase enzymes hydrolyze the long polygalacturonic acid chains found in the smooth regions of pectin.  Interest in this enzyme class continues due to the enzymes ability to macerate tissues of economically important crops and their use in a number of industrial processes.  Very little information is available on the biochemical characteristics of this class of enzymes acting on galacturonic acid oligomers, largely due to the lack of a convenient assay system.  In the work presented here, the thermodynamic parameters of binding as well as some kinetic characteristics of polygalacturonase enzymes acting on galacturonic acid oligomers is demonstrated via isothermal titration calorimetry.  Binding of oligomers varying in chain length is an exothermic process that is enthalpically driven and results in extremely tight binding of the substrate to the enzyme.  Conversely, catalysis of the galacturonic acid oligomers is an endothermic process. The kinetic parameters of galacturonic acid oligomers follow the trend seen with many glycoside hydrolases in that as oligomer length increases, the catalytic rate increases and the affinity of the enzyme for the substrate also increasing.  The result of this work provides further insight into biochemistry of this enzyme class and a means for comparison of polygalacturonase enzymes isolated from different sources.