J. Mol. Biol. (1996) 255, 522–535 Entropy in Bi-substrate Enzymes: Proposed Role of an Alternate Site in Chaperoning Substrate into, and Products out of, Thymidylate Synthase David L. Birdsall*, Janet Finer-Moore and Robert M. Stroud Three steps along the pathway of binding, orientation of substrates and Department of Biochemistry and Biophysics, University of release of products are revealed by X-ray crystallographic structures of California, San Francisco ternary complexes of the wild-type Lactobacillus casei thymidylate synthase CA 94143-0448, USA enzyme. Each complex was formed by diffusion of either the cofactor 5,10-methylene-5,6,7,8-tetrahydrofolate or the folate analog 10-propargyl- 5,8-dideazafolate into binary co-crystals of thymidylate synthase with 2'-deoxyuridine-5'-monophosphate. A two-substrate/enzyme complex is formed where the substrates remain unaltered. The imidazolidine ring is unopened and the pterin of the 5,10-methylene-5,6,7,8-tetrahydrofolate 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 events. The structure of the 2'-deoxyuridine-5'-monophosphate and 10-propargyl-5,8-dideazafolate folate analog complex identifies both ligands in orientations favorable for the initiation of catalysis and resembles the productive complex. A product complex where the ligands have been converted into products of the thymidylate synthase reaction within the crystal, 2'-deoxythymidine-5'- monophosphate and 7,8-dihydrofolate, shows how ligands are situated within the enzyme after catalysis and on the way to product release.  1996 Academic Press Limited *Corresponding author Keywords: thymidylate synthase; X-ray crystallography; enzyme; catalysis Introduction In bi-substrate enzymatic reactions mutual align- ment of substrates against one another prior to catalysis is a major factor in catalytic rate enhancement. Thymidylate synthase (TS) closes around its substrates to sequester and orient them in a major conformational change evoked primarily by binding CH 2 H 4 folate (Kamb et al ., 1992). This change is also dependent on the carboxy terminal COO − group on the enzyme (Perry et al ., 1993). Thus mechanisms by which both substrates are bound and oriented prior to catalysis are under selective pressure for optimization. Yet such mechanisms for binding can rarely be delineated by structural analysis. Three structures of thymidylate synthase elucidate mechanisms of bi-substrate binding and suggest mechanisms by which selective expulsion of products takes place. Intermolecular reactions are catalyzed most effectively by the remarkably high effective concen- tration of reactive groups on the substrates once bound correctly such that catalysis becomes effectively an intramolecular reaction. This contri- bution to enzymatic reaction rate is entropic, and the rate enhancement, relative to the rate at which the same reaction would take place in solution, can be estimated by calculating the entropy change due to fixation of the second substrate against the first (Page and Jencks, 1971; Fersht, 1985). In TS, mutual orientation of substrates is followed by methylene transfer of (CH 2 ) 11 of CH 2 H 4 folate to C5 of dUMP, and finally by hydride transfer from cofactor to the transferred methylene to form the products dTMP and the oxidized form of the cofactor, H 2 folate (Stroud & Finer-Moore, 1993). Conformational changes accompany each step of this reaction, as Abbreviations used: TS, thymidylate synthase; LCTS, Lactobacillus casei TS; ECTS, Escherichia coli TS; CH2THF, CH2H4folate, 5,10-methylene-5,6,7,8- tetrahydrofolate; DHF, H2folate, 7,8-dihydrofolate; CB3717, 10-propargyl-5,8-dideazafolate; PABA, para-amino benzoic acid; dUMP, 2'-deoxyuridine- 5'-monophosphate; dTMP, 2'-deoxythymidine-5'- monophosphate; FdUMP, 5-fluoro-2'deoxyuridine- 5'-monophosphate; DTT, dithiothreitol; rms, root mean square; wt, wild-type. 0022–2836/96/030522–14 $12.00/0  1996 Academic Press Limited