title: Mode of action of site-directed irreversible folate analogue inhibitors of thymidylate synthase.
abstract: 5,8-Dideazafolate analogues are tight binding but not irreversible inhibitors of thymidylate synthase (TS). However, when a chloroacetyl (ClAc) group is substituted at the N10-position of 2-desamino-2-methyl-5,8-dideazafolate (DMDDF), the resulting compound, ClAc-DMDDF, although still a reversible inhibitor (KI = 3.4 x 10(-3) M), gradually inactivates thyA-TS irreversibly at a rate of 0.37 min-1. The corresponding iodoacetyl derivative alkylated the enzyme somewhat slower (k3 = 0.15 min-1 ) than ClAc-DMDDF but was bound more tightly (KI = 1.4 x 10(-5) M), resulting in a second-order rate constant (k3/KI) of inactivation that was 100-fold greater than that of ClAc-DMDDF. A tryptic digest of the ClAc-DMDDF-inactivated enzyme yielded a peptide on HPLC, which revealed that cysteine-146, the residue at the active site that is intimately involved in the catalytic process, had reacted with ClAc-DMDDF to form a covalent bond. This derivative was confirmed indirectly by Edman analysis and more directly by mass spectrometry. Deoxyuridine 5'-monophosphate, a substrate in the catalytic reaction, protected against inactivation. Similar to previously described Lactobacillus casei TS inhibition studies with sulfhydryl reagents [Galivan, J., Noonan, J., and Maley, F. (1977) Arch. Biochem. Biophys. 184, 336-345], the kinetics of inhibition suggested that complete inhibition occurs on reaction of only one of the two active site cysteines, although sequence and amino acid analysis revealed that iodoacetate and ClAc-DMDDF had reacted with both active site cysteines. These studies demonstrate that a sulfhydryl reactive compound that is directed to the folate binding site of TS may diffuse to the active site cysteine, and form a covalent bond with this residue. How this inhibition comes about is suggested in a stereoscopic view of the ligand when modeled to the known crystal structure of Escherichia coli TS.
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, New York 12201, USA.
authors.forename: A P
authors.initials: AP
authors.lastname: Lobo
authors.forename: M G
authors.initials: MG
authors.lastname: Nair
authors.forename: L
authors.initials: L
authors.lastname: Changchien
authors.forename: A
authors.initials: A
authors.lastname: Weichsel
authors.forename: W R
authors.initials: WR
authors.lastname: Montfort
authors.forename: F
authors.initials: F
authors.lastname: Maley
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D018698:Glutamic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D011743:Pyrimidines / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D011799:Quinazolines / Q000378:metabolism / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; C111509:N(10)-chloroacetyl-2-desamino-2-methyl-5,8-dideazafolate; C111510:N(10)-iodoacetyl-2-desamino-2-methyl-5,8-dideazafolate; D011743:Pyrimidines; D011799:Quinazolines; D018698:Glutamic Acid; C031662:CB 3717; D005492:Folic Acid; D013940:Thymidylate Synthase; D003545:Cysteine