Transition state analysis of thymidine hydrolysis by human thymidine phosphorylase

Phillip A. Schwartz, Mathew J. Vetticatt, Vern L. Schramm

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Human thymidine phosphorylase (hTP) is responsible for thymidine (dT) homeostasis, and its action promotes angiogenesis. In the absence of phosphate, hTP catalyzes a slow hydrolytic depyrimidination of dT yielding thymine and 2-deoxyribose (dRib). Its transition state was characterized using multiple kinetic isotope effect (KIE) measurements. Isotopically enriched thymidines were synthesized enzymatically from glucose or (deoxy)ribose, and intrinsic KIEs were used to interpret the transition state structure. KIEs from [1′- 14C]-, [1-15N]-, [1′-3H]-, [2′R-3H]-, [2′S-3H]-, [4′- 3H]-, and [5′-3H]dTs provided values of 1.033 ± 0.002, 1.004 ± 0.002, 1.325 ± 0.003, 1.101 ± 0.004, 1.087 ± 0.005, 1.040 ± 0.003, and 1.033 ± 0.003, respectively. Transition state analysis revealed a stepwise mechanism with a 2-deoxyribocation formed early and a higher energetic barrier for nucleophilic attack of a water molecule on the high energy intermediate. An equilibrium exists between the deoxyribocation and reactants prior to the irreversible nucleophilic attack by water. The results establish activation of the thymine leaving group without requirement for phosphate. A transition state constrained to match the intrinsic KIEs was found using density functional theory. An active site histidine (His116) is implicated as the catalytic base for activation of the water nucleophile at the rate-limiting transition state. The distance between the water nucleophile and the anomeric carbon (rC-O) is predicted to be 2.3 Å at the transition state. The transition state model predicts that deoxyribose adopts a mild 3′-endo conformation during nucleophilic capture. These results differ from the concerted bimolecular mechanism reported for the arsenolytic reaction (Birck, M. R.; Schramm, V. L. J. Am. Chem. Soc. 2004, 126, 2447-2453).

Original languageEnglish (US)
Pages (from-to)13425-13433
Number of pages9
JournalJournal of the American Chemical Society
Volume132
Issue number38
DOIs
StatePublished - Sep 29 2010

Fingerprint

Thymidine Phosphorylase
Thymidine
Hydrolysis
Deoxyribose
Nucleophiles
Thymine
Water
Phosphates
Chemical activation
Ribose
Histidine
Isotopes
Density functional theory
Glucose
Conformations
Catalytic Domain
Homeostasis
Carbon
Molecules
Kinetics

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Transition state analysis of thymidine hydrolysis by human thymidine phosphorylase. / Schwartz, Phillip A.; Vetticatt, Mathew J.; Schramm, Vern L.

In: Journal of the American Chemical Society, Vol. 132, No. 38, 29.09.2010, p. 13425-13433.

Research output: Contribution to journalArticle

Schwartz, Phillip A. ; Vetticatt, Mathew J. ; Schramm, Vern L. / Transition state analysis of thymidine hydrolysis by human thymidine phosphorylase. In: Journal of the American Chemical Society. 2010 ; Vol. 132, No. 38. pp. 13425-13433.
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