P40: Kinetic characteristics of polygalacturonase enzymes hydrolyzing galacturonic acid oligomers using isothermal titration calorimetry

Polygalacturonase enzymes hydrolyze the polygalacturonic acid chains found in pectin.  Interest in polygalacturonase enzymes continues as they are useful in a number of industrial processes and conversely, detrimental, as they are involved in maceration of economically important crops.  While a good deal of biochemical information is available for polygalacturonase enzymes hydrolyzing polygalacturonic acids, little biochemical information exists on the hydrolysis of galacturonic acid oligomers; largely due to the lack of a convenient assay system.  In the work presented here, the kinetic characteristics of polygalacturonases hydrolyzing galacturonic acid oligomers are determined using isothermal titration calorimetry.  Hydrolysis of galacturonic acid oligomers is an endothermic process and essentially enthalpically silent with ΔH_app values of 2 kcal/mol and less than 1 kcal/mol for the exo-and endo- enzymes, respectively.  Kinetic parameters of hydrolysis for two enzymes characterized follow the trend seen with many glycoside hydrolases in that as oligomer length increases, the catalytic rate and the affinity of the enzyme for the substrate increases.  One of the exo-acting polygalacturonase enzymes characterized does not follow this general pattern.  The exo-acting polygalacturonase enzymes are subject to product inhibition with determined K_i values significantly higher than K_m.  This work provides additional insight into biochemistry of this enzyme class and provides a means for comparison of polygalacturonase enzymes isolated from different sources.