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 and release of products are revealed by X-ray crystallographic structures of ternary complexes of the wild-type Lactobacillus casei thymidylate synthase 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.
Birdsall
D L
DL
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448, USA.
Finer-Moore
J
J
Stroud
R M
RM
D001665:Binding Sites; D018360:Crystallography, X-Ray; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D005492:Folic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008958:Models, Molecular; D011487:Protein Conformation; D011799:Quinazolines / Q000378:metabolism; D013763:Tetrahydrofolates / Q000378:metabolism; D013816:Thermodynamics; D013940:Thymidylate Synthase / Q000737:chemistry / Q000378:metabolism*
D003856:Deoxyuracil Nucleotides; D011799:Quinazolines; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; C031662:CB 3717; D005492:Folic Acid; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase