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'-(3)H]-, [1'-(14)C]-, and [5'-(14)C,1,3-(15)N(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(N)D(N) (S(N)2) 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(N)D(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.

Figure 1

Initial rate patterns for the TcUP reaction.

Figure 2

Single-turnover apparent rate constant dependence on TcUP-P_i binary complex concentration. The inset depicts a representative stopped-flow average trace for [TcUP-P_i] = 30 μM.

Figure 3

Rapid kinetics of the TcUP reaction under multiple-turnover conditions demonstrating the absence of burst kinetics in product formation.

Figure 4

Lag phase in the approach to steady-state for uracil formation.

Figure 5

Lag time dependence on the concentration of uridine and (inset) P_i.

Figure 6

pH-rate profiles for TcUP-catalyzed reaction.

Figure 7

Solvent deuterium kinetic isotope effects with P_i and (inset) uridine as varied substrates.

Figure 8

Potential energy surface for uridine phosphorolysis modeled by DFT in the gas phase.

Figure 9

Stick models of stationary-point transition structures for uridine cleavage calculated by DFT in the gas phase with (A) P_i or (B) arsenate as nucleophiles. Carbon atoms are shown in gray, nitrogen in blue, oxygen in red, hydrogen in white, phosphorus in yellow, and arsenic in purple. Bond lengths (in Å) and bond orders (Pauling bond order) are shown for leaving group and nucleophile positions at the calculated transition state.

Scheme 1

Uridine phosphorolysis catalyzed by UP.

Scheme 2

Scheme 3

Scheme 4

Kinetic mechanism proposed for the TcUP reaction.

Scheme 5

Chemical mechanism proposed for TcUP-catalyzed uridine phosphorolysis.