Transition State Analysis of the Arsenolytic Depyrimidination of Thymidine by Human Thymidine Phosphorylase

Abstract

Human thymidine phosphorylase (hTP) is responsible for thymidine (dT) homeostasis, promotes angiogenesis, and is involved in metabolic inactivation of antiproliferative agents that inhibit thymidylate synthase. Understanding its transition state structure is on the path to design transition state analogues. Arsenolysis of dT by hTP permits kinetic isotope effect (KIE) analysis of the reaction by forming thymine and the chemically unstable 2-deoxyribose 1-arsenate. The transition state for the arsenolytic reaction was characterized using multiple KIEs and computational analysis. Transition state analysis revealed a concerted bimolecular (A N D N ) mechanism. A transition state constrained to match the intrinsic KIE values was found using density functional theory [B3LYP/6-31+G*]. An active site histidine is implicated as the catalytic base responsible for activation of the arsenate nucleophile and stabilization of the thymine leaving group during the isotopically sensitive step. At the transition state, the deoxyribose ring exhibits significant oxocarbenium ion character with bond-breaking (r C-N = 2.45 Å) nearly complete but no significant bond-making to the attacking nucleophile (r C-O = 2.95 Å). The transition state model predicts a deoxyribose conformation with a 2′-endo ring geometry. Transition state structure for the slow hydrolytic reaction of hTP involves a stepwise mechanism (Schwartz, P.A., Vetticatt, M.J., Schramm, V.L. (2010) J. Am. Chem. Soc. 132, 13425-13433), in contrast to the concerted mechanism described here for arsenolysis.Human thymidine phosphorylase (hTP) 1 catalyzes the reversible phosphorolytic depyrimidination of thymidine (dT) (1,2):Arsenate is a substrate analogue of phosphate and reduces the reverse reaction to permit transition state analysis by kinetic isotope effects (KIEs) (see commitments to catalysis). The hTP-catalyzed arsenolytic depyrimidination of dT forms an unstable 2-deoxy-α-D-ribose 1-arsenate which undergoes spontaneous hydrolysis (Fig. 1). hTP is involved in dT homeostasis by participating in pyrimidine salvage (3,4).

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

“…TcUP also accepts arsenate as nucleophile, as is observed for other nucleoside phosphorylases. 12,13,16 …”

Uridine Phosphorylase from Trypanosoma cruzi: Kinetic and Chemical Mechanisms

“…The p K a value for the 3-NH group of uridine is 9.2 (42), near the range observed here in the pH-profiles. In the bimolecular transition state model proposed for uridine arsenolysis by TcUP, the leaving group is not immediately protonated at 1-N (11), a common feature of reactions involving pyrimidine nucleoside cleavage (40, 43-45). Thus, the product is an anionic nucleobase following C – N bond breakage and C – O bond formation.…”

“…Deprotonation of 3-NH in the ground state would generate a dianioic uracil at the transition state, a less effective leaving group (Scheme 5). A mono-anionic pyrimidine leaving group has also been implicated in other reactions involving pyrimidine nucleosides N -ribosidic bond breakage (40, 43-45, 47, 48). The S N 2 mechanism proposed here for the TcUP reaction is also found in DFT-based calculations of uridine phosphorolysis in the gas phase.…”

Measuring specificity in multi-substrate/product systems as a tool to investigate selectivity in vivo

“…A scintillation detector with a multi-channel analyzer can record different radiation energy from different radioactive sources. With this technique, a high sensitivity and high accuracy of measurements can be achieved for the detection of various radioactively labeled substrates [48–50]. The study of hydrogen tunneling by using radioactive remote labeling is one example of the application of this technique [51, 52].…”