title: 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.
authors.affiliation: Centro de Pesquisas em Biologia Molecular e Funcional (CPBMF), Instituto Nacional de Ciência e Tecnologia em Tuberculose (INCT-TB), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), 6681/92-A Av. Ipiranga, 90619-900, Porto Alegre, RS, Brazil. luiz.basso@pucrs.br diogenes@pucrs.br.
authors.forename: Candida
authors.initials: C
authors.lastname: Deves
authors.forename: Diana Carolina
authors.initials: DC
authors.lastname: Rostirolla
authors.forename: Leonardo Kras Borges
authors.initials: LK
authors.lastname: Martinelli
authors.forename: Cristiano Valim
authors.initials: CV
authors.lastname: Bizarro
authors.forename: Diogenes Santiago
authors.initials: DS
authors.lastname: Santos
authors.forename: Luiz Augusto
authors.initials: LA
authors.lastname: Basso
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008970:Molecular Weight; D011485:Protein Binding; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D013939:Thymidine Phosphorylase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D008024:Ligands; D011994:Recombinant Proteins; D013939:Thymidine Phosphorylase