Transition state analysis of thymidine phosphorylase from E. coli.

Abstract

Thymidine phosphorylase (E.C. 2.4.2.4) (TP) is an enzyme involved in the reversible conversion of thymidine, deoxyuridine, and their analogs to their respective bases and 2-alpha-D-deoxyribose-1-phosphate. This enzyme is identical to an angiogenic factor, platelet-derived endothelial cell growth factor. TP is expressed at high levels in a wide variety of solid tumors and is known to promote the development of new blood vessels, which are fundamental to tumor growth and metastasis.

The transition state of thymidine phosphorylase has been characterized by kinetic isotope effects, bond-energy bond-order vibrational analysis, and molecular electrostatic potential surface and quantum chemical calculations. Kinetic isotope effects for arsenolysis were measured by liquid chromatography/mass spectrometry for [1'-2H], [2' -2H], [5'-2H], [1'-13C], [2' -13C), and [1-15N] uridine to provide experimental values of 1.144 +/- 0.050, 0.959 +/- 0.012, 0.988 +/- 0.127, 1.013 +/- 0.007, 0.995 +/- 0.002, and 1.027 +/- 0.046 respectively. These kinetic isotope effects were matched to a geometric transition state model selected by bond-energy bond-order vibrational analysis (BEBOVIB-IV program).

The transition state can be described as an SN1 type reaction with oxocarbenium ion character with slight hyperconjugation existing between the C2'-H2' and C1'-N1 bonds, and C4 '-endo and C3'- exo conformation of furanose ring due to protonation of O2 in the uracil ring. Protonation of O2 also assists departure of the uracil. From the BEBOVIB-IV calculations, the 13C isotope effect predicts a bond order of 0.78 for C1'-N 1, indicating an early transition state.

Inhibitor design for thymidine phosphorylase was attempted by incorporating features of the transition state.