Uridine phosphorylase from Trypanosoma cruzi

Kinetic and chemical mechanisms

Rafael G. Silva, Vern L. Schramm

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

The reversible phosphorolysis of uridine to generate uracil and ribose 1-phosphate is catalyzed by uridine phosphorylase and is involved in the pyrimidine salvage pathway. We define the reaction mechanism of uridine phosphorylase from Trypanosoma cruzi by steady-state and pre-steady-state kinetics, pH-rate profiles, kinetic isotope effects from uridine, and solvent deuterium isotope effects. Initial rate and product inhibition patterns suggest a steady-state random kinetic mechanism. Pre-steady-state kinetics indicated no rate-limiting step after formation of the enzyme-products ternary complex, as no burst in product formation is observed. The limiting single-turnover rate constant equals the steady-state turnover number; thus, chemistry is partially or fully rate limiting. Kinetic isotope effects with [1′- 3H]-, [1′- 14C]-, and [5′- 14C,1,3- 15N 2]uridine gave experimental values of α-T(V/K) uridine = 1.063, 14(V/K) uridine = 1.069, and 15,β-15(V/K) uridine = 1.018, in agreement with an A ND N (S N2) mechanism where chemistry contributes significantly to the overall rate-limiting step of the reaction. Density functional theory modeling of the reaction in gas phase supports an A ND N mechanism. Solvent deuterium kinetic isotope effects were unity, indicating that no kinetically significant proton transfer step is involved at the transition state. In this N-ribosyl transferase, proton transfer to neutralize the leaving group is not part of transition state formation, consistent with an enzyme-stabilized anionic uracil as the leaving group. Kinetic analysis as a function of pH indicates one protonated group essential for catalysis and for substrate binding.

Original languageEnglish (US)
Pages (from-to)9158-9166
Number of pages9
JournalBiochemistry
Volume50
Issue number42
DOIs
StatePublished - Oct 25 2011

Fingerprint

Uridine Phosphorylase
Trypanosoma cruzi
Uridine
Kinetics
Isotopes
Proton transfer
Uracil
Deuterium
Protons
Salvaging
Enzymes
Transferases
Catalysis
Density functional theory
Rate constants
Gases

ASJC Scopus subject areas

  • Biochemistry

Cite this

Uridine phosphorylase from Trypanosoma cruzi : Kinetic and chemical mechanisms. / Silva, Rafael G.; Schramm, Vern L.

In: Biochemistry, Vol. 50, No. 42, 25.10.2011, p. 9158-9166.

Research output: Contribution to journalArticle

@article{8871868efe3041ebbea39a89782e09cd,
title = "Uridine phosphorylase from Trypanosoma cruzi: Kinetic and chemical mechanisms",
abstract = "The reversible phosphorolysis of uridine to generate uracil and ribose 1-phosphate is catalyzed by uridine phosphorylase and is involved in the pyrimidine salvage pathway. We define the reaction mechanism of uridine phosphorylase from Trypanosoma cruzi by steady-state and pre-steady-state kinetics, pH-rate profiles, kinetic isotope effects from uridine, and solvent deuterium isotope effects. Initial rate and product inhibition patterns suggest a steady-state random kinetic mechanism. Pre-steady-state kinetics indicated no rate-limiting step after formation of the enzyme-products ternary complex, as no burst in product formation is observed. The limiting single-turnover rate constant equals the steady-state turnover number; thus, chemistry is partially or fully rate limiting. Kinetic isotope effects with [1′- 3H]-, [1′- 14C]-, and [5′- 14C,1,3- 15N 2]uridine gave experimental values of α-T(V/K) uridine = 1.063, 14(V/K) uridine = 1.069, and 15,β-15(V/K) uridine = 1.018, in agreement with an A ND N (S N2) mechanism where chemistry contributes significantly to the overall rate-limiting step of the reaction. Density functional theory modeling of the reaction in gas phase supports an A ND N mechanism. Solvent deuterium kinetic isotope effects were unity, indicating that no kinetically significant proton transfer step is involved at the transition state. In this N-ribosyl transferase, proton transfer to neutralize the leaving group is not part of transition state formation, consistent with an enzyme-stabilized anionic uracil as the leaving group. Kinetic analysis as a function of pH indicates one protonated group essential for catalysis and for substrate binding.",
author = "Silva, {Rafael G.} and Schramm, {Vern L.}",
year = "2011",
month = "10",
day = "25",
doi = "10.1021/bi2013382",
language = "English (US)",
volume = "50",
pages = "9158--9166",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "42",

}

TY - JOUR

T1 - Uridine phosphorylase from Trypanosoma cruzi

T2 - Kinetic and chemical mechanisms

AU - Silva, Rafael G.

AU - Schramm, Vern L.

PY - 2011/10/25

Y1 - 2011/10/25

N2 - The reversible phosphorolysis of uridine to generate uracil and ribose 1-phosphate is catalyzed by uridine phosphorylase and is involved in the pyrimidine salvage pathway. We define the reaction mechanism of uridine phosphorylase from Trypanosoma cruzi by steady-state and pre-steady-state kinetics, pH-rate profiles, kinetic isotope effects from uridine, and solvent deuterium isotope effects. Initial rate and product inhibition patterns suggest a steady-state random kinetic mechanism. Pre-steady-state kinetics indicated no rate-limiting step after formation of the enzyme-products ternary complex, as no burst in product formation is observed. The limiting single-turnover rate constant equals the steady-state turnover number; thus, chemistry is partially or fully rate limiting. Kinetic isotope effects with [1′- 3H]-, [1′- 14C]-, and [5′- 14C,1,3- 15N 2]uridine gave experimental values of α-T(V/K) uridine = 1.063, 14(V/K) uridine = 1.069, and 15,β-15(V/K) uridine = 1.018, in agreement with an A ND N (S N2) mechanism where chemistry contributes significantly to the overall rate-limiting step of the reaction. Density functional theory modeling of the reaction in gas phase supports an A ND N mechanism. Solvent deuterium kinetic isotope effects were unity, indicating that no kinetically significant proton transfer step is involved at the transition state. In this N-ribosyl transferase, proton transfer to neutralize the leaving group is not part of transition state formation, consistent with an enzyme-stabilized anionic uracil as the leaving group. Kinetic analysis as a function of pH indicates one protonated group essential for catalysis and for substrate binding.

AB - The reversible phosphorolysis of uridine to generate uracil and ribose 1-phosphate is catalyzed by uridine phosphorylase and is involved in the pyrimidine salvage pathway. We define the reaction mechanism of uridine phosphorylase from Trypanosoma cruzi by steady-state and pre-steady-state kinetics, pH-rate profiles, kinetic isotope effects from uridine, and solvent deuterium isotope effects. Initial rate and product inhibition patterns suggest a steady-state random kinetic mechanism. Pre-steady-state kinetics indicated no rate-limiting step after formation of the enzyme-products ternary complex, as no burst in product formation is observed. The limiting single-turnover rate constant equals the steady-state turnover number; thus, chemistry is partially or fully rate limiting. Kinetic isotope effects with [1′- 3H]-, [1′- 14C]-, and [5′- 14C,1,3- 15N 2]uridine gave experimental values of α-T(V/K) uridine = 1.063, 14(V/K) uridine = 1.069, and 15,β-15(V/K) uridine = 1.018, in agreement with an A ND N (S N2) mechanism where chemistry contributes significantly to the overall rate-limiting step of the reaction. Density functional theory modeling of the reaction in gas phase supports an A ND N mechanism. Solvent deuterium kinetic isotope effects were unity, indicating that no kinetically significant proton transfer step is involved at the transition state. In this N-ribosyl transferase, proton transfer to neutralize the leaving group is not part of transition state formation, consistent with an enzyme-stabilized anionic uracil as the leaving group. Kinetic analysis as a function of pH indicates one protonated group essential for catalysis and for substrate binding.

UR - http://www.scopus.com/inward/record.url?scp=80054978032&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=80054978032&partnerID=8YFLogxK

U2 - 10.1021/bi2013382

DO - 10.1021/bi2013382

M3 - Article

VL - 50

SP - 9158

EP - 9166

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 42

ER -