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

2021 ◽  
Author(s):  
Felix Kaspar ◽  
Darian S. Wolff ◽  
Peter Neubauer ◽  
Anke Kurreck ◽  
Vickery Arcus
Keyword(s):  
Heat Capacity ◽  
Reaction System ◽  
Heat Capacity Change ◽  
Pyrimidine Nucleoside ◽  
Nucleoside Phosphorylase ◽  
Geobacillus Thermoglucosidasius ◽  
Capacity Change ◽  
Pyrimidine Nucleoside Phosphorylase ◽  
Enzyme Catalyzed Reactions ◽  
Activation Heat

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 <i>Geobacillus thermoglucosidasius</i> across a two-dimensional working space covering 35 °C and 3 pH units with two substrates displaying different pK<sub>a</sub> 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.<br>

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

2021 ◽  
Author(s):  
Felix Kaspar ◽  
Darian S. Wolff ◽  
Peter Neubauer ◽  
Anke Kurreck ◽  
Vickery Arcus
Keyword(s):  
Heat Capacity ◽  
Reaction System ◽  
Heat Capacity Change ◽  
Pyrimidine Nucleoside ◽  
Nucleoside Phosphorylase ◽  
Geobacillus Thermoglucosidasius ◽  
Capacity Change ◽  
Pyrimidine Nucleoside Phosphorylase ◽  
Enzyme Catalyzed Reactions ◽  
Activation Heat

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 <i>Geobacillus thermoglucosidasius</i> across a two-dimensional working space covering 35 °C and 3 pH units with two substrates displaying different pK<sub>a</sub> 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.<br>

scholarly journals Dynamical origins of heat capacity changes in enzyme catalysed reactions

2017 ◽  
Author(s):  
Marc W van der Kamp ◽  
Erica J. Prentice ◽  
Kirsty L. Kraakmann ◽  
Michael Connolly ◽  
Adrian J. Mulholland ◽  
...  
Keyword(s):  
Heat Capacity ◽  
Active Site ◽  
Reaction Rates ◽  
Heat Capacity Change ◽  
Catalysed Reaction ◽  
Capacity Change ◽  
Heat Capacity Changes ◽  
Dynamics Simulations ◽  
Alpha Glucosidase ◽  
Activation Heat

AbstractHeat capacity changes are emerging as essential for explaining the temperature dependence of enzyme-catalysed reaction rates. This has important implications for enzyme kinetics, thermoadaptation and evolution, but the physical basis of these heat capacity changes is unknown. Here we show by a combination of experiment and simulation, for two quite distinct enzymes (dimeric ketosteroid isomerase and monomeric alpha-glucosidase), that the activation heat capacity change for the catalysed reaction can be predicted through atomistic molecular dynamics simulations. The simulations reveal subtle and surprising underlying dynamical changes: tightening of loops around the active site is observed as expected, but crucially, changes in energetic fluctuations are evident across the whole enzyme including important contributions from oligomeric neighbours and domains distal to the active site. This has general implications for understanding enzyme catalysis and demonstrating a direct connection between functionally important microscopic dynamics and macroscopically measurable quantities.

scholarly journals Heat capacity change for ribonuclease A folding

1999 ◽  
Vol 8 (7) ◽  
pp. 1500-1504 ◽  
Author(s):  
C. Nick Pace ◽  
Gerald R. Grimsley ◽  
Susan T. Thomas ◽  
George I. Makhatadze
Keyword(s):  
Heat Capacity ◽  
Ribonuclease A ◽  
Heat Capacity Change ◽  
Capacity Change

scholarly journals Temperature control technology by heat capacity change upon lock and key binding

2010 ◽  
Vol 375 (2) ◽  
pp. 165-169 ◽  
Author(s):  
Ken-ichi Amano ◽  
Daisuke Miyazaki ◽  
Liew Fong Fong ◽  
Paul Hilscher ◽  
Taro Sonobe
Keyword(s):  
Heat Capacity ◽  
Temperature Control ◽  
Heat Capacity Change ◽  
Control Technology ◽  
Capacity Change

scholarly journals The Peculiar Case of the Hyperthermostable Pyrimidine Nucleoside Phosphorylase from Thermus Thermophilus

2020 ◽  
Author(s):  
Felix Kaspar ◽  
Peter Neubauer ◽  
Anke Kurreck
Keyword(s):  
Thermus Thermophilus ◽  
Pyrimidine Nucleoside ◽  
Reaction Conditions ◽  
Nucleoside Phosphorylase ◽  
Pyrimidine Nucleosides ◽  
Low Concentrations ◽  
Pyrimidine Nucleoside Phosphorylase ◽  
Total Turnover ◽  
Nucleoside Phosphorylases ◽  
Peculiar Case

The poor solubility of many nucleoside and nucleobases in aqueous solution demands harsh reaction conditions (base, heat, cosolvent) in nucleoside phosphorylase-catalyzed processes to facilitate substrate loading beyond the low millimolar range. This, in turn, requires enzymes which withstand these conditions. Herein we report that the pyrimidine nucleoside phosphorylase from <i>Thermus thermophilus</i> is active over an exceptionally broad pH (4-10), temperature (up to 100 °C) and cosolvent space (up to 80% (v/v) non-aqueous medium) and displays tremendous stability under harsh reaction conditions with predicted total turnover numbers of more than 10<sup>6</sup> for various pyrimidine nucleosides. However, its use as a biocatalyst for preparative applications is critically limited due to its inhibition by nucleoside substrates at low concentrations, which is unprecedented among non-specific pyrimidine nucleoside phosphorylases.<br>

scholarly journals Characterization of pyrimidine nucleoside phosphorylase of Mycoplasma hyorhinis: implications for the clinical efficacy of nucleoside analogues

2012 ◽  
Vol 445 (1) ◽  
pp. 113-123 ◽  
Author(s):  
Johan Vande Voorde ◽  
Federico Gago ◽  
Kristof Vrancken ◽  
Sandra Liekens ◽  
Jan Balzarini
Keyword(s):  
Nucleoside Analogues ◽  
Kinetic Properties ◽  
Virus Infections ◽  
Pyrimidine Nucleoside ◽  
Mycoplasma Hyorhinis ◽  
Nucleoside Phosphorylase ◽  
Secondary Infections ◽  
Pyrimidine Nucleoside Phosphorylase ◽  
In Silico Analyses

In the present paper we demonstrate that the cytostatic and antiviral activity of pyrimidine nucleoside analogues is markedly decreased by a Mycoplasma hyorhinis infection and show that the phosphorolytic activity of the mycoplasmas is responsible for this. Since mycoplasmas are (i) an important cause of secondary infections in immunocompromised (e.g. HIV infected) patients and (ii) known to preferentially colonize tumour tissue in cancer patients, catabolic mycoplasma enzymes may compromise efficient chemotherapy of virus infections and cancer. In the genome of M. hyorhinis, a TP (thymidine phosphorylase) gene has been annotated. This gene was cloned, expressed in Escherichia coli and kinetically characterized. Whereas the mycoplasma TP efficiently catalyses the phosphorolysis of thymidine (Km=473 μM) and deoxyuridine (Km=578 μM), it prefers uridine (Km=92 μM) as a substrate. Our kinetic data and sequence analysis revealed that the annotated M. hyorhinis TP belongs to the NP (nucleoside phosphorylase)-II class PyNPs (pyrimidine NPs), and is distinct from the NP-II class TP and NP-I class UPs (uridine phosphorylases). M. hyorhinis PyNP also markedly differs from TP and UP in its substrate specificity towards therapeutic nucleoside analogues and susceptibility to clinically relevant drugs. Several kinetic properties of mycoplasma PyNP were explained by in silico analyses.

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