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,1,3-15N2]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.

UDP-glucose Dehydrogenase: The First-step Oxidation Is an NAD+-dependent Bimolecular Nucleophilic Substitution Reaction (SN2)

“…To the best of our knowledge, this NAD + -dependent S N 2 mechanism has not been reported before, although many other enzymes are known to involve an S N 2 reaction, e.g. , haloalkane dehalogenase 42-45, uridine diphosphate glucuronosyltransferase 46, sorbitol dehydrogenase 47, and uridine phosphorylase 48-50. These S N 2 reactions are however independent of the cofactor NAD + .…”

The Peculiar Case of the Hyper‐thermostable Pyrimidine Nucleoside Phosphorylase from Thermus thermophilus**

“…[13]) to other thymidine and pyrimidine nucleoside phosphorylases concerning the active site residues and overall protein structure. Although substrate inhibition is known for uridine phosphorylases, [28,30] this is, to the best of our knowledge, the first reported example of a pyrimidine nucleoside phosphorylase being competitively substrate/product‐inhibited. However, we doubt that this inhibition of Tt PyNP holds any physiological significance, as the intracellular concentrations of nucleosides and their bases are typically in the low‐micromolar range, which is more than two orders of magnitude lower than the concentrations necessary to effect significant inhibition of Tt PyNP.…”

The molecular structure of Schistosoma mansoni PNP isoform 2 provides insights into the nucleoside selectivity of PNPs

“…Moreover, a proposed catalytic mechanism for Trypanosoma cruzi uridine phosphorylase [ 51 ] seems an adequate mechanism to describe the mechanism for cytidine cleavage by Sm PNP2. Under the original model a protonated group, possibly uridine NH3, is essential for catalysis, its deprotonation generates a dianionic uracil a less effective leaving group.…”