Submitted to: Thermochimica Acta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/27/2011
Publication Date: 11/2/2011
Citation: Mertens, J.A., Hector, R.E., Bowman, M.J. 2012. Subsite binding energies of an exo-polygalacturonase using isothermal titration calorimetry. Thermochimica Acta. 527:219-222.
Interpretive Summary: Meeting future fuel and specialty chemical needs will require the use of agricultural biomass. To be fully utilized, biomass needs to be broken down by chemical pre-treatments and enzymes into simple sugars. Rhizopus oryzae makes enzymes called polygalacturonases that are very effective in the conversion of pectin, a component of biomass, into simple sugars. Industrial interest in this enzyme is strong due to the use of this enzyme in clarifying fruit juices, textile production, and potential in the conversion of biomass to simple sugars. In this work we have determined the energetic properties of an exo-polygalacturonase binding to the complex sugars. This work aids in a greater understanding of how this enzyme works to break down more complex sugars and paves the way for development and engineering of more effective enzymes.
Technical Abstract: Thermodynamic parameters for binding of a series of galacturonic acid oligomers to an exo-polygalacturonase, RPG16 from Rhizopus oryzae, were determined by isothermal titration calorimetry. Binding of oligomers varying in chain length from two to five galacturonic acid residues is an exothermic process that is enthalpically driven and results in extremely tight binding of the substrate to RPG16. Binding energies in combination with prior biochemical data suggests that RPG16 has the potential for five subsites, -1 to +4, with the greatest contribution to binding energies arising from subsite -1/+1. While the enthalpic contribution to binding decreases substantially for subsites +2 to +4, beneficial entropic effects occur in subsites +3 and +4 leading to increased total free energy as the length of oligomer increases. This information will be useful for additional studies in determining the binding contributions of specific amino acids with mutant enzymes.