Properties of phosphorylated thymidylate synthase

Abstract

Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent.

Mouse thymidylate synthase does not show the inactive conformation, observed for the human enzyme

“…Considering strong influences of posttranslational modifications on kinetics and inhibition of TS-catalyzed reaction [9,26,27] and resulting wide variability of properties of the enzyme preparations of the same specific origin [2,9,27], a simple comparison of mTS and hTS properties would not answer the former question. However, Gibson et al [28] obtained a mutant R163K of the human enzyme and found its catalytic activity to be higher than that of the parental hTS.…”

“…The enzyme preparation was separated into phosphorylated and non-phosphorylated fractions as previously described [12]. Only the non-phosphorylated fraction was used for crystallization.…”

Phosphorylation of thymidylate synthase affects slow-binding inhibition by 5-fluoro-dUMP and N4-hydroxy-dCMP

“…ref. 16), only the upper one shows the presence of a phosphate group, as detected in lane 2, causing lower electrophoretic mobility of the phosphorylated subunit). Assuming the presence of a fraction of TS molecules phosphorylated on both subunits, it was too small to be detected.…”

“…Alternatively, rare enzyme molecules that underwent phosphorylation in bacterial cells on both subunits could be lost in the process of MOAC, the loss also remaining undetected. The two TS fractions have been previously characterized by comparing their 31 P NMR spectra and found to contain unmodified and histidine-phosphorylated enzymes, 16 respectively. The latter was evidenced by the phosphorylated fraction spectrum containing a resonance at 2.14 ppm, belonging to inorganic phosphate, and two upfield shifted singlet peaks at À7.39 ppm and À9.87 ppm, with the singlet resonances in the negative spectrum region assigned to reflect N-phosphorylated histidine species (3-pHis and 1-pHis, respectively).…”

“…The latter was confirmed by the acidlabile character of both resonances. 16 H]dUMP tritium release activity assay. Each inhibitor, when preincubated with TS, in the presence of meTHF, caused time-dependent inactivation of the enzyme, consistent with the behavior as a slow-binding inhibitor.…”