The kinetic mechanism of Human Thymidine Phosphorylase – a molecular target for cancer drug development

Abstract

Human Thymidine Phosphorylase (HTP), also known as the platelet-derived endothelial cell growth factor (PD-ECGF) or gliostatin, catalyzes the reversible phosphorolysis of thymidine (dThd) to thymine and 2-deoxy-α-d-ribose-1-phosphate (2dR1P). HTP is a key enzyme in the pyrimidine salvage pathway involved in dThd homeostasis in cells. HTP is a target for anticancer drug development as its enzymatic activity promotes angiogenesis. Here, we describe cloning, expression, and purification to homogeneity of recombinant TYMP-encoded HTP. Peptide fingerprinting and the molecular mass value of the homogenous protein confirmed its identity as HTP assessed by mass spectrometry. Size exclusion chromatography showed that HTP is a dimer in solution. Kinetic studies revealed that HTP displayed substrate inhibition for dThd. Initial velocity and isothermal titration calorimetry (ITC) studies suggest that HTP catalysis follows a rapid-equilibrium random bi-bi kinetic mechanism. ITC measurements also showed that dThd and Pi binding are favorable processes. The pH-rate profiles indicated that maximal enzyme activity was achieved at low pH values. Functional groups with apparent pK values of 5.2 and 9.0 are involved in dThd binding and groups with pK values of 6.1 and 7.8 are involved in phosphate binding.

Genetic correlation between multiple myeloma and chronic lymphocytic leukaemia provides evidence for shared aetiology

“…Variants within this locus were enriched in H3K4me3 chromatin marks in both CD38- B cells and inflammatory macrophages. TYMP (alias ECGF1) encodes thymidine phosphorylase, which is often overexpressed in tumours and has been linked to angiogenesis 54,55 . A detailed study on this gene has implicated TYMP in the development of lytic bone lesions in MM, via a mechanism involving activation of PI3K/Akt signalling and increased DNMT3A expression resulting in hypermethylation of RUNX2 , osterix, and IRF8 56 .…”

Granulocyte-Colony Stimulating Factor Related Pathways Tested on an Endometrial Ex-Vivo Model

“…Thymidine Phosphorylase (TYMP), also known as the platelet-derived endothelial cell growth factor (PD-ECGF) or gliostatin, is a key angiogenesis promoting enzyme [20] as well as a cell migration promoter, especially by modulating integrin expression [21] . Its presence is also described in the endometrium [22] .…”

Engineering of the Recombinant Expression and PEGylation Efficiency of the Therapeutic Enzyme Human Thymidine Phosphorylase

“…Available information deposited in public research databases (e.g., NCBI and Uniprot) suggests that HsTP harbors a N'-terminal 10residue long pro-peptide (Uniprot entry P19971) which is cleaved during a post-translational modification and processing step, thereby yielding the final mature polypeptide chain. While the biological role of this pro-peptide, as well as its impact on the enzyme's biochemical features remain largely unclear, all prior studies focusing on the biochemical characterization of HsTP used the putatively mature version of the protein with the first ten residues at the N'-terminal site truncated (Schwartz et al, 2010;Deves et al, 2014). Accordingly, we designed a HsTP gene carrying a His 6 -tag at the N'-terminus, codon-optimized for E. coli expression, and devoid of the first ten residues corresponding to the pro-peptide sequence, cloned into pET28-a plasmid (construct termed HsTP 199 , Figure 1D) and tested its recombinant expression in the E. coli BL21 (DE3) strain.…”

“…HsTP does not show any significant amino acid sequence similarity against any of the HsUP enzymes whereas HsUP1 and HsUP2 share 66% sequence identity. HsTP is a highly specific N'-ribosyl phosphorylase displaying k cat /K M values against dThd and dUrd in the range of 10 5 M −1 s −1 and 10 4 M −1 s −1 respectively (Deves et al, 2014;Schwartz et al, 2010), whereas HsUP enzymes exhibit higher specificity against uridine (Urd) (k cat /K M ∼10 5 M −1 s −1 ) and to a lesser extent towards dUrd and dThd (k cat /K M ∼10 3 M −1 s −1 ) (Liu et al, 1998;Renck et al, 2010). Thymidine phosphorylases from lower organisms like Bacillus stearothermophilus (BsTP) and Escherichia coli (EcTP) exhibit similar fold and belong to the same family (family II) of nucleoside phosphorylases with HsTP, while their amino acid sequence identity is ∼40%.…”