Thymidine phosphorylase from Escherichia coli: Tight-binding inhibitors as enzyme active-site titrants

Abstract

Thymidine phosphorylase (EC 2.4.2.4) catalyses the reversible phosphorolysis of pyrimidine 2′-deoxynucleosides, forming 2-deoxyribose-1-phosphate and pyrimidine. 5-Chloro-6-(2-imino-pyrrolidin-1-yl)methyl-uracil hydrochloride (TPI, 1) and its 5-bromo analogue (2), 6-(2-amino-imidazol-1-yl)methyl-5-bromo-uracil (3) and its 5-chloro analogue (4) act as tight-binding stoichiometric inhibitors of recombinant E. coli thymidine phosphorylase, and thus can be used as the first active-site titrants for it using either thymidine or 5-nitro-2′-deoxyuridine as substrate.

Keywords::

• Stoichiometric inhibition
• inhibitors
• angiogenesis
• anticancer drugs
• transition state inhibitor
• thymidine phosphorylase

Acknowledgements

We are grateful to Dr Simon Barry of AstraZeneca PLC (UK) for helpful discussions, access to the X-ray data for the human enzyme and for supplies of human TP as a glutathione transferase construct, and to the Libyan Cultural Affairs Office (London) for financial support (AG).