Mechanism of action of selected biomacromolecules and their constituents, with the aid of absorption and emission spectroscopy, and kinetic methods, including time-resolved techniques

Enzyme-ligand interactions (L+E ⇋ EL) lead to a remarkable increase of the mean lifetime of ligand emission (Stoychev et al., 2003), and to fluorescence resonance energy transfer (FRET) between tyrosine residues and ligand moieties (Kierdaszuk et al., 2000). This enable to identify tautomeric form of formycin B (selective non-substrate inhibitor) preferred by E. coli PNP (Kierdaszuk et al., 2000, Kierdaszuk, 2002). Hence, results obtained by emission spectroscopy of enzyme-ligand complexes in solution removed ambiguities found in roentgenographic studies of the enzyme-ligand complex in solid state (Koellner et al., JMB 280, 153-166 (1998) (see Project A for further details).

People involved
Borys Kierdaszuk
Krzysztof Krawiec

Collaboration with (in Department of Biophysics)
Anna Modrak-Wójcik

Further collaboration
Dr. Matthias Bochtler (Joint MPG-PAN Junior Research Group, International Institute of Molecular and Cell Biology, Warsaw, Poland)

Prof. Gert Dandanell (Institute of Molecular Biology, Department of Biological Chemistry, Copenhagen, Denmark)

Prof. Staffan Eriksson (Department of Veterinary Medicinal Chemistry, Biomedical Center, Uppsala, Sweden)

Dr Maciej Garstka (Department of Metabolism Physiology, Institute of Biochemistry, University of Warsaw, Head: Prof. Bryła)

Prof. Joseph R. Lakowicz (Center for Fluorescence Spectroscopy, University of Maryland at Baltimore, Baltimore, USA)

Prof. Birgitte Munch-Petersen (Department of life Sciences and Chemistry, Roskilde University, Roskilde, Denmark)

Dr hab. Maciej Nowak and Dr hab. Bolesław Kozankiewicz (Institute of Physics, Polish Academy of Sciences, Warsaw, Head: Prof. Jerzy Prochorow).

Dr Jarosław Poznański (Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw)

Prof. Rudolf Rigler (Department of Medical Biophysics, Karolinska Institute, Stockholm, Sweden)

Laboratory for Emission Spectroscopy of Biomolecules (LESBM)
Laboratory enable steady-state and time-resolved fluorescence and phosphorescence studies at different temperature and oxygen content, and includes

  • Laser source of excitation performed by Ti:Sapphire laser pumped by argon-ion laser; Detection with time-correlated single-photon counting, with resolution in the picosecond-nanosecond range;
  • Nanosecond time-resolved spectrofluorimeter (IBH System 5000) with flash-lamp;
  • Home made phosphorimeter, which enable steady-sate and time resolved phosphorescence measurements with resolution from microseconds to seconds;
  • Nitrogen cryostat for temperatures 80-300 K, useful for emission detection in glassy solutions;

mechanism of enzyme action,
enzyme-ligand interactions,
absorption and emission (fluorescence, phosphorescence) spectroscopy,
time-resolved spectroscopy, emission probes,
enzyme kinetics,
purine nucleoside phosphorylases,
deoxyribonucleoside kinases,
chloroplasts of higher plants

Research interests

Projekt A: Mechanism of action of purine nucleoside phosphorylases (PNP) based on spectroscopic, chromatographic, crystallographic and kinetic studies of enzyme-ligand complexes

Purine nucleoside phosphorylase (PNP) catalyses the cleavage of the glycosidic bond of ribo- and deoxyribonucleosides of guanine and hypoxanthine in higher organisms, as well as of adenine in some prokaryotes (e.g. E. coli), in the presence of inorganic orthophosphate (Pi), as a second substrate. For the natural substrates the reaction is reversible, as follows:

<center>purine β-nucleoside + Pi ⇋ purine base + α-D-pentose-1-phosphate</center>

Equilibrium of the in vitro reaction is shifted in favor of nucleoside synthesis. However, in vivo phosphorolysis is highly favored over synthesis, due to its coupling with two additional enzymatic reactions, viz. oxidation and phosphoribosylation of the purine bases by xanthine oxidase and hypoxanthine-guanine phosphoribosyltransferase, respectively.

PNP has been considered as primary target for selective immunosuppressive agents, and for potentiation of the antitumour and antiviral activities of therapeutically active nucleoside analogues. Differences in specificity between PNPs from human and viral or bacterial sources have been profited from for development of so-called tumour-directed gene therapy for treatment of cancer.

There are two PNPs in E. coli, i.e. PNP-I and PNP-II, the products of two different genes DeoD and xapA, respectively.

Important contribution in studies of the mechanism of action of these enzymes was made by our group (Włodarczyk & Kierdaszuk, 2003; Kierdaszuk et al., 1997, 2000; Kierdaszuk, 2002; Stoychev et al., 2001, 2002) using emission spectroscopy and enzyme kinetics for further clarification of enzyme-ligand interactions. We have shown for the first time the remarkable preference of E. coli PNP-I towards N(2)-H tautomeric form of formycin A and B independently on the inorganic orthophosphate, as well as fluorescence resonance energy transfer between tyrosine residues of protein and base moiety of ligand (see also Figure, top-left side). In contrast, structural preferences of the enzyme towards amino or imino form of N(6)-methylformycin A appear to be dependent on the presence and absence of phosphate (Kierdaszuk et al., 2000; Kierdaszuk, 2002), respectively. Although interaction of phosphate with PNP-I is very complex (Kierdaszuk et al., 1997), it reflects bimodal enzyme kinetics and shades a new light on the possible mechanism of its dual role in this system.

We have also shown for the first time molecular bases for differences in substrate specificity between E. coli PNP-I and mammalian PNP including E. coli PNP-II towards xanthosine and xanthine (Stoychev et al., 2002). Our explanation is based on the prototropic equilibria (tautomeric, proton dissociation-association), which was usually omitted and/or ignored in previously reported studies on substrate specificity of PNP as well as of many other enzyme systems, where these compounds exist as primary substrates and/or intermediates, e.g. in biosynthesis of caffeine. We proposed new mode of enzyme-xanthosine and enzyme-xanthine binding, which appear to be very important in the catalytic reaction (Stoychev et al., 2002).

Projekt B: Striking substrate/inhibitor specificities of four human, and the multifunctional Drosophila, 2'-deoxyribonucleoside kinases

Deoxyribonucleoside kinases belong to the pathway of re-usage of nucleosides - salvage pathway, also named in Polish "rescue pathway" or "reserve pathway", and catalyze the phosphorylation of a 2'-deoxyribonucleosides (dN) to 2'-deoxyribonucleside-5'-monophosphate (dNMP) in the presence of a nucloeside-5'-triphosphate phosphate donor (NTP). In mammalian cells there are four deoxyribonucleoside kinases, namely cytosolic deoxycytidine kinase (dCK), cytosolic thymidine kinase (TK1), mitochondrial thymidine kinase (TK2) and mitochondrial deoxyguanosine kinase (dGK).

Some organisms (e.g. Drosophila melanogaster) contain one multifunctional deoxyribonucleoside kinase (dNK) with much broader substrate specificity then human kinases. Detail knowledge on factors responsible for these differences will enable application of the gene of dNK (or other kinases) in so called tumour-directed gene therapy.

While the cytosolic route is to complement the de novo synthesis of dNTPs (substrates for biosynthesis of DNA), activities of deoxyribonucleoside kinases in mitochondria probably are crucial for mitochondrial DNA replication and repair.

On the other hand, a large number of animal viruses encode their own TKs that possess very broad substrate specificities and are able to phosphorylate many nucleoside analogues that can not be accepted by cellular kinases. For example the Herpes simplex virus family codes for deoxyribonucleoside kinase (viral TK) with a broad specificity, which selectively activates the most efficient antiviral drug (Acyclovir) known to date. The differences in the substrate recognition between viral and cellular deoxyribonucleoside kinases actually are the molecular basis for the efficacy of many antiviral compounds against several types of viral infections.

For example, deoxycytidine kinase catalyzes the phosphorylation of a 2'-deoxycytydine to 2'-deoxycytydine-5'-monophosphate in the presence of a nucloeside-5'-triphosphate donor (NTP), not only ATP but also UTP (Krawiec et al., 1995) as well as adenosine-2'(3')-deoxy-3'(2')-triphosphates (Krawiec et al., 1998, 2003), and tripolyphosphate (Krawiec et al., 2003); and, despite its name, is also responsible for phosphorylation of purine 2'-deoxyribonucleosides.

We have shown for the first time that all deoxyribonucleoside kinases (or, more precisely, enzyme activities) may by divided into two groups: (a) TK1, TK2, dNK and dCK (but only with dAdo as acceptor), with restricted donor specificity, are significantly active only with adenosine-3'-deoxy-2'-triphosphate; (b) dCK (with dCyd as acceptor) and dGK, with relaxed donor specificity, which accept all adenosine-2'(3')-deoxy-3'(2')-triphosphates and their analogues (Krawiec et al., 1998, 2003) at a level comparable with, or even superior to, that for ATP. Furthermore, we show that interactions of the enzymes with phosphate donors depend on phosphate acceptor, which together with their substrate properties exhibit important feature of the mechanism of action of these enzymes in both in vitro and in vivo.

Projekt C: Mechanism of function of thylakoid membranes in higher plants

Absorption and fluorescence spectroscopy, CD, and confocal microscopy were employed in studies of spectral properties of thylakoid membranes isolated from higher plants, at different concentrations of magnesium ions and detergents, which affect spectra of chloroplasts and quantum yield of photosynthesis. Measurements are usually performed at different concentrations of oxygen, which were controlled using saturation with nitrogen or argon and/or enzymatic depletion of oxygen directly in the solutions. Results obtained thus far were interpreted using hypothesis about possible effect of magnesium and detergent on the membrane structure, specifically reflected in the resonance energy transfer (RET) phenomena. The latter were observed by relative changes in fluorescence intensity in fluorescence emission (650-750 nm) and excitation spectra (400-520 nm).

Selected publications

  1. Włodarczyk J. and Kierdaszuk B., Interpretation of fluorescence decays using a power-like model. Biophysical Journal, 85(1), in press (2003)
  2. Krawiec K., Kierdaszuk B. and Shugar D., Inorganic tripolyphosphate (PPPi) as a phosphate donor for human deoxyribonucleoside kinases. Biochemical and Biophysical Research Communications 301, 192-197 (2003)
  3. Poznański J., Kierdaszuk B. and Shugar D., Structural properties of the neutral and monoanioic forms of xanthosine, highly relevant to their substrate properties with various enzyme systems. Nucleosides, Nucleotides & Nucleic Acids, 22, 249-263 (2003)
  4. Krawiec K., Kierdaszuk B., Kalinichenko E. N., Rubinova E. B., Mikhailopulo I. A., Eriksson S., Munch-Petersen B. and Shugar D., Striking ability of adenosine-2'(3')-deoxy-3'(2')-triphosphates, and related analogues, to replace ATP as phosphate donor for all human, and Drosphila melanogaster, deoxyribonucleoside kinases. Nucleosides, Nucleotides & Nucleic Acids, 22, 153-173 (2003)
  5. Kierdaszuk B., Emission spectroscopy of complex formation between Escherichia coli purine nucleoside phosphorylase (PNP) and identified tautomeric species of formycin inhibitors resolves ambiguities found in crystallographic studies. Fluorescence Spectroscopy, Imaging and Probes 2, 277-296 (2002)
  6. Stoychev G., Kierdaszuk B. and Shugar D., Xanthosine and xanthine: Substrate properties with purine nucleoside phosphorylases, and relevance to other enzyme systems. European Journal of Biochemistry 269, 4048-4057 (2002)
  7. Stoychev G., Kierdaszuk B. and Shugar D., Interaction of E. coli purine nucleoside phosphorylase (PNP) with the cationic and zwitterionic forms of the fluorescent substrate N(7)-methylguanosine. Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology, 1544, 74-88 (2001)
  8. Kierdaszuk B., Modrak-Wójcik A., Wierzchowski J. and Shugar D., Formycin A and its N-methyl analogues, specific inhibitors of E. coli purine nucleoside phosphorylase (PNP): induced tautomeric shifts on binding to enzyme, and enzyme-ligand fluorescence resonance energy transfer. Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology 1476, 109-128 (2000)
  9. Stepanenko T., Lapinski L., Sobolewski A.L., Nowak M.J. and Kierdaszuk B., Photochemical syn-anti isomerisation reaction in 1-methyl-N4-hydroxycytosine. An experimental matrix isolation and theoretical density functional theory study. Journal of Physical Chemistry, 104, 9459-9466 (2000)
  10. Kierdaszuk B., Krawiec K., Kazimierczuk Z., Jacobsson U., Johansson N.G., Munch Petersen B., Eriksson S. and Shugar D., Substrate/inhibitor specificities of human deoxycytidine kinase (dCK) and thymidine kinase (TK1 and TK2) towards the sugar moiety of nucleosides, including O'-alkyl analogues. Nucleosides & Nucleotides 18, 1883-1903 (1999)
  11. Kierdaszuk B., Krawiec K., Kazimierczuk Z., Jacobsson U., Johansson N.G., Munch-Petersen B., Eriksson S. and Shugar D., Substrate/inhibitor specificities of human deoxycytidine kinase (dCK) and thymidine kinase (TK1 and TK2). Advances in Experimental Medicine and Biology 431, 623-627 (1998)
  12. Niedzwiecka-Kornas A., Kierdaszuk B., Stolarski R. and Shugar D. Tautomerism, acid-base properties and conformation of methylated analogues of the promutagenic N4-hydroxycytosine. Biophysical Chemistry 71, 87-98 (1998)
  13. Krawiec K., Kierdaszuk B., Kalinichenko E.N., Mikhailopulo I.A. and Shugar D., Unusual nucleoside triphosphate donors for nucleoside kinases: 3'-deoxyadenosine-2'-triphosphate and 2'-deoxyadenosine-3'-triphosphate. Acta Biochimica Polonica 45, 87-94 (1998)
  14. Wieczorek Z., Zdanowski K., Chlebicka L., Stępiński J., Jankowska M., Kierdaszuk B., Temeriusz A., Darżynkiewicz E. and Stolarski R., Fluorescence and NMR studies of intramolecular stacking of mRNA cap-analogues. Biochimica et Biophysica Acta 1354, 145-152 (1997)
  15. Kierdaszuk B., Gryczynski I. and Lakowicz J.R., Two-photon induced fluorescence of proteins. In Topics in Fluorescence Spectroscopy, Volume 5: Nonlinear and Two-Photon-Induced Fluorescence, (Lakowicz, J. Ed.) New York: Plenum Publishing Co., pp. 187-209 (1997)
  16. Kierdaszuk B., Modrak-Wojcik A. and Shugar D., Binding of phosphate and sulfate anions by purine nucleoside phosphorylase from E. coli: Ligand-dependent quenching of enzyme intrinsic fluorescence. Biophysical Chemistry 63, 107-118 (1997)
  17. Birnbaum K., Kierdaszuk B. and Shugar D., Crystal structure of 1,3,5-trimethyl-N4-hydroxycytosine, and its relevance to the mechanism of hydroxylamine mutagenesis. Nucleosides & Nucleotides 15, 1805-1819 (1996)
  18. Kierdaszuk B., Malak H. Gryczynski I., Calis P. and Lakowicz J.R., Fluorescence of reduced nicotinamides using one- and two-photon excitation. Biophysical Chemistry 62, 1-13 (1996)
  19. Lakowicz J.R., Kierdaszuk B., Malak H. and Gryczynski I., Fluorescence of horse liver alcohol dehydrogenase using one- and two-photon excitation. Journal of Fluorescence 6, 51-59 (1996)
  20. Lakowicz J.R., Kierdaszuk B., Calis P., Malak H. and Gryczynski I., Fluorescence anisotropy of tyrosine using one- and two-photon excitation. Biophysical Chemistry 56, 263-271 (1995)
  21. Krawiec K., Kierdaszuk B., Eriksson S., Munch-Petersen B. and Shugar D., Nucleoside triphosphate donors for nucleoside kinases: donor properties of UTP with human deoxycytidine kinase. Biochemical and Biophysical Research Communications 216, 42-48 (1995)
  22. Krawiec K., Kierdaszuk B. and Shugar D., 5'-Substituted 2'-Deoxycytidine as non-substrate Inhibitors of human deoxycytidine kinase. Nucleosides & Nucleotides 14, 495-499 (1995)
  23. Bretner M., Balinska M., Krawiec K., Kierdaszuk B., Shugar D. and Kulikowski T., Synthesis and Biological Activity of 5-Fluoro-2-thiocytosine Nucleosides. Nucleosides & Nucleotides 14, 657-660 (1995)
  24. Kierdaszuk B., Gryczynski I., Modrak-Wojcik A., Bzowska A., Shugar D. and Lakowicz J.R., Fluorescence of tyrosine and tryptophan in proteins using one- and two-photon excitation. Photochemistry and Photobiology 61, 319-324 (1995)

Supported by

Polish State Committee for Scientific Research (KBN): 3 projects in 1992-2000

Howard Hughes Medical Institute (USA): 5-years grant

Foundation for Polish Science (FNP) in the frame of FASTKIN-97 program

International collaborations: 2 collaborative projects with: Prof. Birgitte Munch-Petersen and Prof. Gert Dandanell (Denmark), and Prof. Rudolf Rigler and Prof. Staffan Eriksson (Sweden)

European Science Foundation: participation in the European Project "Femtochemistry and femtobiology ULTRA"