A synergistic effect of phosphate, pH and Phe159 substitution on the formycin A association to the E. coli purine nucleoside phosphorylase
| Autor: | Joanna Cieśla, Borys Kierdaszuk, Małgorzata Prokopowicz |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Absorption spectroscopy Stereochemistry Phenylalanine Purine nucleoside phosphorylase Biochemistry Phosphates Active center 03 medical and health sciences chemistry.chemical_compound Escherichia coli chemistry.chemical_classification Quenching (fluorescence) Wild type General Medicine Hydrogen-Ion Concentration Phosphate Dissociation constant 030104 developmental biology Enzyme Amino Acid Substitution Purine-Nucleoside Phosphorylase chemistry Mutation Formycins Protein Binding |
| Zdroj: | Biochimie. 148:80-86 |
| ISSN: | 0300-9084 |
| DOI: | 10.1016/j.biochi.2018.02.012 |
| Popis: | A steady-state absorption and emission spectroscopy was used to create a comprehensive work and to study the interaction of the wild type Escherichia coli purine nucleoside phosphorylase and its mutants, PNPF159Y and PNPF159A, with a potent E. coli PNP inhibitor - formycin A. The absorption and emission spectra were recorded in the presence and absence of the phosphate at the 50 mM concentration. From the collected sets of data dissociation constants (Kd), apparent dissociation constants (Kapp) and Hill's coefficients (h) were calculated. Additionally, the temperature dependence of the enzymes emission quenching at two temperatures, 10 °C and 25 °C, was examined. To verify the calculations, total difference absorption spectra were computed for all types of the complexes. A prominent quenching of the PNPF159Y emission indicates a complex formation, with the strongest association in the phosphate buffer, pH 7, relative to the wild type enzyme. On the other hand, results testify to a deterioration of the interactions in the E. coli PNP/PNPF159Y and formycin A complexes in the presence of the phosphate, pH 8.3. Moreover, data obtained for the PNPF159A-FA complexes confirm a weak association of the FA to the mutant's active center. |
| Databáze: | OpenAIRE |
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