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 mu M 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 mu M, 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 mu M for RPG15 and RPG16, respectively. RPG15 exhibited greater affinity for (GalpA)(3) with a K (m) of 9.2 mu M 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.