THE ADDITIVITY OF SUBSTRATE FRAGMENTS IN ENZYME-LIGAND BINDING

Background: Enzymes have evolved to recognise their target substrates with exquisite selectivity and specificity. Whether fragments of the s ubstrate perhaps never available to the evolving enzyme - are bound in the same manner as the parent substrate addresses the fundamental bas is of specificity. An understanding of the relative contributions of i ndividual portions of ligand molecules to the enzyme-binding interacti on may offer considerable insight into the principles of substrate rec ognition, Results: We report 12 crystal structures of Escherichia coli thymidylate synthase in complexes with available fragments of the sub strate (dUMP), both with and without the presence of a cofactor analog ue. The structures display considerable fidelity of binding mode and i nteractions. These complexes reveal several interesting features: the cofactor analogue enhances the localisation of substrate and substrate fragments near the reactive thiol; the ribose moiety reduces local di sorder through additional specific enzyme-ligand interactions; the pyr imidine has multiple roles, ranging from stereospecificity to mechanis tic competence; and the glycosidic linkage has an important role in th e formation of a covalent attachment between substrate and enzyme. Con clusions: The requirements of ligand-protein binding can be understood in terms of the binding of separate fragments of the ligand. Fragment s which are subsystems of the natural substrate for the enzyme confer specific contributions to the binding affinity, orientation or electro statics of the enzymatic mechanism. This ligand-binding analysis provi des a complementary method to the more prevalent approaches utilising site-directed mutagenesis. In addition, these observations suggest a m odular approach for rational drug design utilising chemical fragments.