Kinetic Properties of Two Rhizopus Exo-polygalacturonase Enzymes Hydrolyzing Galacturonic Acid Oligomers Using Isothermal Titration Calorimetry |
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| Authors: | Jeffrey A Mertens |
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| Institution: | 1. Bioenergy Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, US Department of Agriculture, 1815 North University Street, Peoria, IL, 61604, USA
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| Abstract: | The kinetic characteristics of two Rhizopus oryzae exo-polygalacturonases acting on galacturonic acid oligomers (GalpA) were determined using isothermal titration calorimetry (ITC). RPG15 hydrolyzing (GalpA)2 demonstrated a K m of 55 μM and k cat of 10.3 s?1 while RPG16 was shown to have greater affinity for (GalpA)2 with a K m of 16 μM, but lesser catalytic activity with a k cat of 3.9 s?1. Both enzymes were inhibited by the product, galacturonic acid, with app K i values of 886 and 501 μM for RPG15 and RPG16, respectively. RPG15 exhibited greater affinity for (GalpA)3 with a K m of 9.2 μM and a similar k cat at 10.7 s?1 relative to (GalpA)2. Catalytic constants for RPG16 hydrolyzing (GalpA)3 could not be determined; however, single-injection ITC assays suggest a distinct preference and catalytic rate for (GalpA)3 relative to (GalpA)2. Thermodynamic parameters of a series of galacturonic acid oligomers binding to RPG15 were determined and exhibited some distinct differences from RPG16 binding thermodynamics, providing potential clues to the differing kinetic characteristics of the two exo-polygalacturonase enzymes. |
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