The kinetic mechanism of human uridine phosphorylase 1: Towards the development of enzyme inhibitors for cancer chemotherapy.
Uridine phosphorylase (UP) is a key enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate (R1P). The human UP type 1 (hUP1) is a molecular target for the design of inhibitors intended to boost endogenous uridine levels to rescue normal tissues from the toxicity of fluoropyrimidine nucleoside chemotherapeutic agents, such as capecitabine and 5-fluorouracil. Here, we describe a method to obtain homogeneous recombinant hUP1, and present initial velocity, product inhibition, and equilibrium binding data. These results suggest that hUP1 catalyzes uridine phosphorolysis by a steady-state ordered bi bi kinetic mechanism, in which inorganic phosphate binds first followed by the binding of uridine, and uracil dissociates first, followed by R1P release. Fluorescence titration at equilibrium showed cooperative binding of either P(i) or R1P binding to hUP1. Amino acid residues involved in either catalysis or substrate binding were proposed based on pH-rate profiles.
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), 90619-900 Porto Alegre, RS, Brazil.
Daiana
D
Renck
Rodrigo G
RG
Ducati
Mario S
MS
Palma
Diógenes S
DS
Santos
Luiz A
LA
Basso
D002384:Catalysis; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D009369:Neoplasms / Q000188:drug therapy*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012267:Ribosemonophosphates / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D014543:Uridine Phosphorylase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D012267:Ribosemonophosphates; C031154:ribose 1-phosphate; D014543:Uridine Phosphorylase