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

02 March 2021, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Enzyme-catalyzed reactions sometimes display curvature in their Eyring plots in the absence of denaturation, indicative of a change in activation heat capacity. However, pH and (de)protonation effects 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 °C and 3 pH units with two substrates displaying different pKa values. Our analysis revealed the presence of a measurable activation heat capacity change 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 which has a minor influence on the heat capacity change but conveys a significant kinetic effect by lowering 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 condition can affect rate, affinity and thermodynamic phenomena independently of one another.


Heat Capacity

Supplementary materials

Kaspar GtPyNP 2021 Supplementary Info

Supplementary weblinks


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