pH-Independent Heat Capacity Changes during Phosphorolysis Catalyzed by the Pyrimidine Nucleoside Phosphorylase from Geobacillus thermoglucosidasius

Enzyme-catalyzed reactions sometimes display curvature in their Eyring plots in the absence of denaturation, indicative of a change in activation heat capacity. However, the effects of pH and (de)protonation on this phenomenon have remained unexplored. Herein, we report a kinetic characterization of the thermophilic pyrimidine nucleoside phosphorylase from Geobacillus thermoglucosidasius across a two-dimensional working space covering 35 degrees C and 3 pH units with two substrates displaying different pK(a) values. Our analysis revealed the presence of a measurable activation heat capacity change Delta C-p double dagger. in this reaction system, which showed no significant dependence on medium pH or substrate charge. Our results further describe the remarkable effects of a single halide substitution that has a minor influence on Delta C-p double dagger. but conveys a significant kinetic effect by decreasing the activation enthalpy, causing a >10-fold rate increase. Collectively, our results present an important piece in the understanding of enzymatic systems across multidimensional working spaces where the choice of reaction conditions can affect the rate, affinity, and thermodynamic phenomena independently of one another.

Automated summary

In this study, a kinetic characterization of a specific enzyme system revealed a measurable change in activation heat capacity in the reaction system, with stability across different medium pH and substrate charge. Furthermore, the results showed that a single halide substitution had minimal impact on ΔC-p double dagger but significantly decreased the activation enthalpy, resulting in a substantial increase in reaction rate.