Search results

1

Molecular mechanism of thymidylate synthase-catalyzed reaction and interaction of the enzyme with 2- and/or 4-substituted analogues of dUMP and 5-fluoro-dUMP

100%

Rode W., Leś A.

Acta Biochimica Polonica

|

1996

|

vol. 43

|

issue 1

133-142

2

Phosphorylation of basic amino acid residues in proteins: important but easily missed

81%

Cieśla J., Frączyk T., Rode W.

Acta Biochimica Polonica

|

2011

|

vol. 58

|

issue 2

137-148

EN

Reversible phosphorylation is the most widespread posttranslational protein modification, playing regulatory role in almost every aspect of cell life. The majority of protein phosphorylation research has been focused on serine, threonine and tyrosine that form acid-stable phosphomonoesters. However, protein histidine, arginine and lysine residues also may undergo phosphorylation to yield acid-labile phosphoramidates, most often remaining undetected in conventional studies of protein phosphorylation. It has become increasingly evident that acid-labile protein phosphorylations play important roles in signal transduction and other regulatory processes. Beside acting as high-energy intermediates in the transfer of the phosphoryl group from donor to acceptor molecules, phosphohistidines have been found so far in histone H4, heterotrimeric G proteins, ion channel KCa3.1, annexin 1, P-selectin and myelin basic protein, as well as in recombinant thymidylate synthase expressed in bacterial cells. Phosphoarginines occur in histone H3, myelin basic protein and capsidic protein VP12 of granulosis virus, whereas phospholysine in histone H1. This overview of the current knowledge on phosphorylation of protein basic amino-acid residues takes into consideration its proved or possible roles in cell functioning. Specific requirements of studies on acid-labile protein phosphorylation are also indicated.

3

Altered mouse leukemia L1210 thymidylate synthase, associated with cell resistance to 5-fluoro-dUrd, is not mutated but rather reflects posttranslational modification

71%

Cieśla J., Frączyk T., Zieliński Z., Sikora J., Rode W.

Acta Biochimica Polonica

|

2006

|

vol. 53

|

issue 1

189-198

EN

Thymidylate synthase purified from 5-fluoro-dUrd-resistant mouse leukemia L1210 cells (TSr) was less sensitive to slow-binding inhibition by 5-fluoro-dUMP than the enzyme from the parental cells (TSp), both enzyme forms differing also in sensitivity to several other dump analogues, apparent molecular weights of monomer and dimer, and temperature dependence of the catalyzed reaction. Direct sequencing of products obtained from RT-PCR, performed on total RNA isolated from the parental and 5-fluoro-dUrd-resistant cells, proved both nucleotide sequences to be identical to the mouse thymidylate synthase coding sequence published earlier (NCBI protein database access no. NP_067263). This suggests that the altered properties of TSr are caused by a factor different than protein mutation, presumably posttranslational modification. As a possibility of rat thymidylate synthase phosphorylation has been recently demonstrated (Samsonoff et al. (1997) J Biol Chem 272: 13281), the mouse enzyme amino-acid sequence was analysed, revealing several potential phosphorylation sites. In order to test possible influence of the protein phosphorylation state on enzymatic properties, endogenous TSp and TSr were purified in the presence of inhibitors of phosphatases. Although both enzyme forms were phosphorylated, as shown by electrophoretical separation followed by phosphoprotein detection, the extent of phosphorylation was apparently similar. However, the same two purified enzyme preparations, compared to the corresponding preparations purified in the absence of phosphatase inhibitors, showed certain properties, including sensitivity to the slow-binding inhibition by FdUMP, altered. Thus properties dependence on phosphorylation was indicated.

4

Thymidylate synthase in the development of Hymenolepis diminutu

71%

Cieśla J., Zieliński Z., Machnicka B., Rode W.

Acta Biochimica Polonica

|

1987

|

vol. 34

|

issue 3

291-298

5

Methionhie synthase assay based on coupling with thymidylate synthase reaction

70%

Rode W., Balińska M.

Acta Biochimica Polonica

|

1985

|

vol. 32

|

issue 2

163-167

6

Specificity of thymidylate synthase inactivation by 4,5-bisubstituted dUMP analogues

70%

Rode W.

Acta Biochimica Polonica

|

1993

|

vol. 40

|

issue 3

363-368

7

Acyclic analogues of 5-fluoro-dUMP and 5-fluoro-2'-deoxyuridine: Synthesis and inhibition of thymidylate synthase and tumour cell growth

62%

Felczak K., Gołos B., Dzik J. M., Rode W., Bretner M., Shugar D., Kulikowski T.

Acta Biochimica Polonica

|

1998

|

vol. 45

|

issue 1

75-82

8

Sulfamide antifolates inhibiting thymidylate synthase: synthesis, enzyme inhibition and cytotoxicity.

62%

Pawełczak K., Makowski M., Kempny M., Dzik J. M., Gołos B., Rode W., Rzeszotarska B.

|

vol. 49

|

issue 2

407-420

EN

Synthesis and biological evaluation are described of seven new analogues (3-9) of two potent thymidylate synthase inhibitors, 10-propargyl-5,8-dideazafolate (1) and its 2-methyl-2-deamino congener ICI 198583 (2). While the new compunds 3 and 4 were analogues of 1 and 2, respectively, containing a p-aminobenzenesulfonyl residue in place of the p-aminobenzoic acid residue, the remaining 5 new compounds were analogues of 4 with the L-glutamic acid residue replaced by glycine (5), L-valine (6), L-alanine (7), L-phenylglycine (8) or L-norvaline (9). The new analogues were tested as inhibitors of thymidylate synthases isolated from tumour (Ehrlich carcinoma), parasite (Hymenolepis diminuta) and normal tissue (regenerating rat liver) and found to be weaker inhibitors than the parent 10-propargyl-5,8-dideazafolic acid. Selected new analogues, tested as inhibitors of growth of mouse leukemia L 5178Y cells, were less potent than the parent 10-propargyl-5,8-dideazafolic acid. Substitution of the glutamyl residue in compound 4 with l-norvaline (9) resulted in only a 5-fold stronger thymidylate synthase inhibitor, but a 40-fold weaker cell growth inhibitor.

9

The effect of Arg209 to Lys mutation in mouse thymidylate synthase.

62%

Cieśla J., Gołos B., Wałajtys-Rode E., Jagielska E., Płucienniczak A., Rode W.

|

vol. 49

|

issue 3

651-658

EN

Mouse thymidylate synthase R209K (a mutation corresponding to R218K in Lactobacillus casei), overexpressed in thymidylate synthase-deficient Escherichia coli strain, was poorly soluble and with only feeble enzyme activity. The mutated protein, incubated with FdUMP and N5,10-methylenetetrahydrofolate, did not form a complex stable under conditions of SDS/polyacrylamide gel electrophoresis. The reaction catalyzed by the R209K enzyme (studied in a crude extract), compared to that catalyzed by purified wild-type recombinant mouse thymidylate synthase, showed the Km value for dUMP 571-fold higher and Vmax value over 50-fold (assuming that the mutated enzyme constituted 20% of total crude extract protein) lower. Thus the ratios kcat, R209K/kcat, 'wild' and (kcat, R209K/Km, R209KdUMP)/( kcat, 'wild'/Km, 'wild'dUMP) were 0.019 and 0.000032, respectively, documenting that mouse thymidylate synthase R209, similar to the corresponding L. casei R218, is essential for both dUMP binding and enzyme reaction.

10

An isotopic assay of dUTPase activity based on coupling with thymidylate synthase

60%

Gołos B., Rode W.

Acta Biochimica Polonica

|

1999

|

vol. 46

|

issue 3

837-840

11

Synthesis and biological activity of Nα-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenyl-acetyl]-L-glutamic acid.

52%

Kusakiewicz-Dawid A., Bugaj M., Dzik J. M., Gołos B., Wińska P., Pawełczak K., Rzeszotarska B., Rode W.

|

vol. 49

|

issue 1

197-202

EN

2-Deamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) is a potent inhibitor of thymidylate synthase. Its analogue, Nα-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenylacetyl]-L-glutamic acid, containing p-aminophenylacetic acid residue substituting p-aminobenzoic acid residue, was synthesized. The new analogue exhibited a moderately potent thymidylate synthase inhibition, of linear mixed type vs. the cofactor, N5,10 -methylenetetrahydrofolate. The Ki value of 0.34 μM, determined with a purified recombinant rat hepatoma enzyme, was about 30-fold higher than that reported for inhibition of thymidylate synthase from mouse leukemia L1210 cells by ICI 198583 (Hughes et al., 1990, J. Med. Chem. 33, 3060). Growth of mouse leukemia L5178Y cells was inhibited by the analogue (IC50 = 1.26 μM) 180-fold weaker than by ICI 198583 (IC50 = 6.9 μM).

Refine search results

Molecular mechanism of thymidylate synthase-catalyzed reaction and interaction of the enzyme with 2- and/or 4-substituted analogues of dUMP and 5-fluoro-dUMP

Phosphorylation of basic amino acid residues in proteins: important but easily missed

Altered mouse leukemia L1210 thymidylate synthase, associated with cell resistance to 5-fluoro-dUrd, is not mutated but rather reflects posttranslational modification

Thymidylate synthase in the development of Hymenolepis diminutu

Methionhie synthase assay based on coupling with thymidylate synthase reaction

Specificity of thymidylate synthase inactivation by 4,5-bisubstituted dUMP analogues

Acyclic analogues of 5-fluoro-dUMP and 5-fluoro-2'-deoxyuridine: Synthesis and inhibition of thymidylate synthase and tumour cell growth

Sulfamide antifolates inhibiting thymidylate synthase: synthesis, enzyme inhibition and cytotoxicity.

The effect of Arg209 to Lys mutation in mouse thymidylate synthase.

An isotopic assay of dUTPase activity based on coupling with thymidylate synthase

Synthesis and biological activity of Nα-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenyl-acetyl]-L-glutamic acid.