Inactivation of thymidylate synthetase by a novel mechanism-based enzyme inhibitor: 1-(β-D-2′-deoxyribofuranosyl)8-azapurin-2-one 5′-monophosphate

Incubation of thymidylate synthetase from Lactobacillus casei with the novel substrate analog 1-(2′-deoxyribosyl)8-azapurin-2-one 5′-monophosphate (I) resulted in a time dependent, irreversible, loss of enzyme activity (k_i=1.4 min^-1; K_i=1.6×10^-5 M). The presence or absence of the cofactor 5,10-CH₂-tetrahydrofolate did not influence the inhibition, whereas the substrate deoxyuridylate afforded protection against inactivation of the enzyme by I. The destruction of the electrophilic center at position 6 by reduction to the dihydro derivative transformed I into a reversible competitive inhibitor (K_i=1.2×10^-4 M). Mechanistic considerations suggest that I acts as an enzyme generated, covalently bound, transition state analog.