The kinetic mechanism of human uridine phosphorylase 1: Towards the development of enzyme inhibitors for cancer chemotherapy

Renck, Daiana; Ducati, Rodrigo G.; Palma, Mário Sérgio; Santos, Daniel Silas Veras dos; Basso, Luiz Augusto

doi:10.1016/j.abb.2010.03.004

Cited by 18 publications

(34 citation statements)

References 36 publications

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“…A steady-state random mechanism is also compatible with isotope-trapping experiments and kinetic isotope effect measurements with arsenate as the nucleophile, which ruled out any ordered mechanism with the nucleophile binding first and a steady-state ordered mechanism with uridine binding first to the enzyme (11). These results differ from the steady-state ordered mechanisms reported for human type 1 (6) and rat (23) UPs in which P i is the first substrate to bind. Rapid-equilibrium random mechanisms have been proposed for UPs from E. coli (4) and Lactobacillus casei (24).…”

Section: Resultscontrasting

confidence: 99%

Uridine Phosphorylase from Trypanosoma cruzi: Kinetic and Chemical Mechanisms

Silva

Schramm

2011

Biochemistry

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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,1,3-15N2]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 ANDN (SN2) 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 ANDN 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.

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Section: Resultscontrasting

confidence: 99%

Uridine Phosphorylase from Trypanosoma cruzi: Kinetic and Chemical Mechanisms

Silva

Schramm

2011

Biochemistry

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“…32 The specificity of TcUP for uridine and 2′-deoxyuridine is comparable, evidenced by the similar values of the specificity constants ( k cat K M ). Thus, the ribosyl 2′-OH group is not essential for substrate binding or catalysis.…”

Section: Resultsmentioning

confidence: 90%

Transition-State Analysis of Trypanosoma cruzi Uridine Phosphorylase-Catalyzed Arsenolysis of Uridine

et al. 2011

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Uridine phosphorylase catalyzes the reversible phosphorolysis of uridine and 2′-deoxyuridine to generate uracil and (2-deoxy)ribose 1-phosphate, an important step in the pyrimidine salvage pathway. The coding sequence annotated as a putative nucleoside phosphorylase in the Trypanosoma cruzi genome was overexpressed in Escherichia coli, purified to homogeneity, and shown to be a homodimeric uridine phosphorylase, with similar specificity for uridine and 2′-deoxyuridine, and undetectable activity towards thymidine and purine nucleosides. Competitive kinetic isotope effects (KIEs) were measured and corrected for a forward commitment factor using arsenate as the nucleophile. The intrinsic KIEs are: 1′-14C = 1.103, 1,3-15N2 = 1.034, 3-15N = 1.004, 1-15N = 1.030, 1′-3H = 1.132, 2′-2H = 1.086 and 5′-3H2 = 1.041 for this reaction. Density functional theory was employed to quantitatively interpret the KIEs in terms of transition state structure and geometry. Matching of experimental KIEs to proposed transition state structures suggests an almost synchronous, SN2-like transition state model, in which the ribosyl moiety possesses significant bond order to both nucleophile and leaving group. Natural bond orbital analysis allowed a comparison of the charge distribution pattern between the ground state and the transition state model.

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“…It is suggested that Pi binds to the free enzyme followed by uridine. Uracil then leaves the ternary complex, followed by dissociation of ribose-1-phosphate [19].…”

Section: Resultsmentioning

confidence: 99%

Pan-Pathway Based Interaction Profiling of FDA-Approved Nucleoside and Nucleobase Analogs with Enzymes of the Human Nucleotide Metabolism

et al. 2012

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To identify interactions a nucleoside analog library (NAL) consisting of 45 FDA-approved nucleoside analogs was screened against 23 enzymes of the human nucleotide metabolism using a thermal shift assay. The method was validated with deoxycytidine kinase; eight interactions known from the literature were detected and five additional interactions were revealed after the addition of ATP, the second substrate. The NAL screening gave relatively few significant hits, supporting a low rate of “off target effects.” However, unexpected ligands were identified for two catabolic enzymes guanine deaminase (GDA) and uridine phosphorylase 1 (UPP1). An acyclic guanosine prodrug analog, valaciclovir, was shown to stabilize GDA to the same degree as the natural substrate, guanine, with a ΔTagg around 7°C. Aciclovir, penciclovir, ganciclovir, thioguanine and mercaptopurine were also identified as ligands for GDA. The crystal structure of GDA with valaciclovir bound in the active site was determined, revealing the binding of the long unbranched chain of valaciclovir in the active site of the enzyme. Several ligands were identified for UPP1: vidarabine, an antiviral nucleoside analog, as well as trifluridine, idoxuridine, floxuridine, zidovudine, telbivudine, fluorouracil and thioguanine caused concentration-dependent stabilization of UPP1. A kinetic study of UPP1 with vidarabine revealed that vidarabine was a mixed-type competitive inhibitor with the natural substrate uridine. The unexpected ligands identified for UPP1 and GDA imply further metabolic consequences for these nucleoside analogs, which could also serve as a starting point for future drug design.

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The kinetic mechanism of human uridine phosphorylase 1: Towards the development of enzyme inhibitors for cancer chemotherapy

Cited by 18 publications

References 36 publications

Uridine Phosphorylase from Trypanosoma cruzi: Kinetic and Chemical Mechanisms

Uridine Phosphorylase from Trypanosoma cruzi: Kinetic and Chemical Mechanisms

Transition-State Analysis of Trypanosoma cruzi Uridine Phosphorylase-Catalyzed Arsenolysis of Uridine

Pan-Pathway Based Interaction Profiling of FDA-Approved Nucleoside and Nucleobase Analogs with Enzymes of the Human Nucleotide Metabolism

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