The kinetic mechanism of human uridine phosphorylase 1

Towards the development of enzyme inhibitors for cancer chemotherapy

Daiana Renck, Rodrigo G. Ducati, Mario S. Palma, Diógenes S. Santos, Luiz A. Basso

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

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 Pi or R1P binding to hUP1. Amino acid residues involved in either catalysis or substrate binding were proposed based on pH-rate profiles.

Original languageEnglish (US)
Pages (from-to)35-42
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume497
Issue number1-2
DOIs
StatePublished - May 2010
Externally publishedYes

Fingerprint

Uridine Phosphorylase
Chemotherapy
Uridine
Enzyme Inhibitors
Drug Therapy
Kinetics
Uracil
Neoplasms
Salvaging
Catalysis
Titration
Nucleosides
Fluorouracil
Toxicity
Fluorescence
Phosphates
Tissue
Amino Acids
Substrates
Enzymes

Keywords

  • Cancer chemotherapy
  • Fluorescence spectroscopy
  • Initial velocity
  • PH-rate profiles
  • Product inhibition
  • Uridine phosphorylase kinetic mechanism

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

The kinetic mechanism of human uridine phosphorylase 1 : Towards the development of enzyme inhibitors for cancer chemotherapy. / Renck, Daiana; Ducati, Rodrigo G.; Palma, Mario S.; Santos, Diógenes S.; Basso, Luiz A.

In: Archives of Biochemistry and Biophysics, Vol. 497, No. 1-2, 05.2010, p. 35-42.

Research output: Contribution to journalArticle

@article{b5b47c57e56c4bca9ce558de9381e927,
title = "The kinetic mechanism of human uridine phosphorylase 1: Towards the development of enzyme inhibitors for cancer chemotherapy",
abstract = "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 Pi or R1P binding to hUP1. Amino acid residues involved in either catalysis or substrate binding were proposed based on pH-rate profiles.",
keywords = "Cancer chemotherapy, Fluorescence spectroscopy, Initial velocity, PH-rate profiles, Product inhibition, Uridine phosphorylase kinetic mechanism",
author = "Daiana Renck and Ducati, {Rodrigo G.} and Palma, {Mario S.} and Santos, {Di{\'o}genes S.} and Basso, {Luiz A.}",
year = "2010",
month = "5",
doi = "10.1016/j.abb.2010.03.004",
language = "English (US)",
volume = "497",
pages = "35--42",
journal = "Archives of Biochemistry and Biophysics",
issn = "0003-9861",
publisher = "Academic Press Inc.",
number = "1-2",

}

TY - JOUR

T1 - The kinetic mechanism of human uridine phosphorylase 1

T2 - Towards the development of enzyme inhibitors for cancer chemotherapy

AU - Renck, Daiana

AU - Ducati, Rodrigo G.

AU - Palma, Mario S.

AU - Santos, Diógenes S.

AU - Basso, Luiz A.

PY - 2010/5

Y1 - 2010/5

N2 - 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 Pi or R1P binding to hUP1. Amino acid residues involved in either catalysis or substrate binding were proposed based on pH-rate profiles.

AB - 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 Pi or R1P binding to hUP1. Amino acid residues involved in either catalysis or substrate binding were proposed based on pH-rate profiles.

KW - Cancer chemotherapy

KW - Fluorescence spectroscopy

KW - Initial velocity

KW - PH-rate profiles

KW - Product inhibition

KW - Uridine phosphorylase kinetic mechanism

UR - http://www.scopus.com/inward/record.url?scp=77952554598&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77952554598&partnerID=8YFLogxK

U2 - 10.1016/j.abb.2010.03.004

DO - 10.1016/j.abb.2010.03.004

M3 - Article

VL - 497

SP - 35

EP - 42

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

IS - 1-2

ER -