ENTROPY IN BI-SUBSTRATE ENZYMES - PROPOSED ROLE OF AN ALTERNATE SITE IN CHAPERONING SUBSTRATE INTO, AND PRODUCTS OUT OF, THYMIDYLATE SYNTHASE

Three steps along the pathway of binding, orientation of substrates an d release of products are revealed by X-ray crystallographic structure s of ternary complexes of the wild-type Lactobacillus casei thymidylat e synthase enzyme. Each complex was formed by diffusion of either the cofactor 5,10-methylene-5,6,7,8-tetrahdrofolate or the folate analog 1 0-propargyl-5,8-dideazafolate into binary co-crystals of thymidylate s ynthase with 2'-deoxyuridine-5'-monophosphate. A two-substrate/enzyme complex is formed where the substrates remain unaltered. The imidazoli dine ring is unopened and the pterin of the 5,10-methyl ene-5,6,7,8 -t etrahydrofolate cofactor binds at an unproductive ''alternate'' site. We propose that the presence of the pterin at this site may represent an initial interaction with the enzyme that precedes all catalytic eve nts. The structure of the 2'-deoxyuridine-5'-monophosphate and 10-prop argyl-5,8-dideazafolate folate analog complex identifies both ligands in orientations favorable for the initiation of catalysis and resemble s the productive complex. A product complex where the ligands have bee n converted into products of the thymidylate synthase reaction within the crystal, 2'-deoxy thymidine-5-monophosphate and 7,8-dihydrofolate, shows how ligands are situated within the enzyme after catalysis and on the way to product release. (C) 1996 Academic Press Limited