A synergistic effect of phosphate, pH and Phe159 substitution on the formycin A association to the E. coli purine nucleoside phosphorylase

Małgorzata Prokopowicz , Joanna Cieśla , Borys Kierdaszuk

Abstract

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.
Author Małgorzata Prokopowicz
Małgorzata Prokopowicz,,
-
, Joanna Cieśla (FC / IBC / DDTB)
Joanna Cieśla,,
- Department Of Drug Technology And Biotechnology
, Borys Kierdaszuk
Borys Kierdaszuk,,
-
Journal seriesBiochimie, ISSN 0300-9084, (A 25 pkt)
Issue year2018
Vol148
Pages80-86
Publication size in sheets0.5
Keywords in EnglishAbsorption; Association; E.coli PNP mutants; E.coli purine nucleoside phosphorylase (PNPWT); Emission; Formycin A
DOIDOI:10.1016/j.biochi.2018.02.012
URL https://www.ncbi.nlm.nih.gov/pubmed/29499297
Languageen angielski
File
wdpb_publikacje_pliki_plik_publikacja_3447_org.pdf 1.14 MB
Score (nominal)30
ScoreMinisterial score = 25.0, 23-07-2018, ArticleFromJournal
Ministerial score (2013-2016) = 30.0, 23-07-2018, ArticleFromJournal
Publication indicators WoS Impact Factor: 2016 = 3.112 (2) - 2016=3.156 (5)
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