version date: 1 December 2006 (DHF). Dihydrofolate reductase and serine hydroxymethyltransferase are required by the cell to transform consumed coenzyme back to the active n No-methylenetetrahydrofolate form The whole pathway is illustrated in Fig. 3 dumP FdUMP dTMP Thymidylate Ha folate synthase 7, 8-Dihydrofolate Glycine Wl serine nadPH hydroxymethyl- Dihydrofolate eductase/Methotrexate folate Fig 3 Cell thymidilate biosynthetic pathway involving the enzymes TS, DHFR, and serine hydroxymethyltransferase Based on background information, the student draws the initial steps of the catalytic mechanism as shown in Scheme 1 [8]. By this mechanism, the reaction proceeds by the catalytic Michael addition of a cysteine residue( Cys 146)to C6 of dUMP, generating the dUMP enolate able to attack either the cyclic or open form of methy lenetetrahydrofolate Apparently, the preferred way is the much more reactive Mannich basic form of the N iminium ion of methylenetetrahydrofolate, allowing the formation of a dUMP and cofactor containing ternary complex, covalently binding the enzyme. Deprotonation of the C5 proton of dUMP, assisted by a basic tyrosine residue (Tyr 94), is the next step originating tetrahydrofolate(THF) and a C5 exocyclic methylene group containing dUMP, still bound to the enzyme. Finally, the C5 exocyclic methylene group may be reduced by the transfer of an <www.iupac.org/publications/cd/medicinalchemistry
11 (DHF). Dihydrofolate reductase and serine hydroxymethyltransferase are required by the cell to transform consumed coenzyme back to the active N5 ,N10-methylenetetrahydrofolate form. The whole pathway is illustrated in Fig. 3. Fig. 3 Cell thymidilate biosynthetic pathway involving the enzymes TS, DHFR, and serine hydroxymethyltransferase. Based on background information, the student draws the initial steps of the catalytic mechanism as shown in Scheme 1 [8]. By this mechanism, the reaction proceeds by the catalytic Michael addition of a cysteine residue (Cys 146) to C6 of dUMP, generating the dUMP enolate, able to attack either the cyclic or open form of methylenetetrahydrofolate. Apparently, the preferred way is the much more reactive Mannich basic form of the N5 - iminium ion of methylenetetrahydrofolate, allowing the formation of a dUMP and cofactor containing ternary complex, covalently binding the enzyme. Deprotonation of the C5 proton of dUMP, assisted by a basic tyrosine residue (Tyr 94), is the next step originating tetrahydrofolate (THF) and a C5 exocyclic methylene group containing dUMP, still bound to the enzyme. Finally, the C5 exocyclic methylene group may be reduced by the transfer of an Thymidylate synthase PLP 7,8-Dihydrofolate FdUMP dUMP Glycine Serine Methotrexate Aminopterin Serine hydroxymethyl- transferase Dihydrofolate reductase N5 , N10, Methylene H4 folate H4 folate dTMP NADPH + H+ NADP + <www.iupac.org/publications/cd/medicinal_chemistry/> version date: 1 December 2006
version date: 1 December 2006 hydride from THF C6 to give a dTMP-enzyme enolate and dihydrofolate(DHF), probably due to the proper co-enzyme C6 hydrogen orbital alignment with the exo-methylene group (Scheme 1) N5N10 N-methylene- tetrahydrofolate o H2 NHR dUMP-enolate methylene -dUMP-enzyme Enzyme NHR dTmP. dTMP R:-(p-C6Hs)C(O)NHCH(CO H)CH2CH CO2H Scheme 1 Proposed mechanism for the thymidylate synthase-catalyzed reaction <www.iupac.org/publications/cd/medicinalchemistry/> 12
12 hydride from THF C6 to give a dTMP-enzyme enolate and dihydrofolate (DHF), probably due to the proper co-enzyme C6 hydrogen orbital alignment with the exo-methylene group (Scheme 1). HN N N H N O H2N H H2C NR H B MeN N H O H O O OH O3PO S Cys146 HN N N H N O H2N H CH2 NHR HN N H H O O O OH O3PO S Cys HN N N H N O H2N H CH2 NHR HN N H H O O O OH O3PO S Cys B HN N N H N O H2N H CH2 NHR HN N H O O- O OH O3PO S Cys B HN N N H H N O H2N H CH2 NHR HN N H O O O OH O3PO S Cys H HN CH3 N O H O- O OH O3PO S Cys HN N N H N O H2N NHR HN CH3 O N H O O OH O3PO - S Cys = = = = = Enzyme Enzyme Enzyme Enzyme Enzyme = Enzyme + Enzyme + dUMP N5 ,N10-methylenetetrahydrofolate dUMP-enolate ternary complex THF 5-methylene-dUMP-enzyme dTMP-enzyme complex DHF dTMP 10 5 N5 -methylenetetrahydrofolate R: -(p-C6H5)C(O)NHCH(CO2H)CH2CH2CO2H 5 6 11 Tyr94 Enzyme 6 5 Scheme 1 Proposed mechanism for the thymidylate synthase-catalyzed reaction. <www.iupac.org/publications/cd/medicinal_chemistry/> version date: 1 December 2006
version date: 1 December 2006 Tutorial A Protein Explorer contact analysis of the enzyme-substrate complex, PDB entry lkzi from E. coli, revealed both the ligand binding place and the binding interactions between substrate and cofactor [9]. Indeed, long-range recognition factors between drug and receptor represented by hydrogen bond may be assessed and inferred in terms of electrostatic character and steric orientation. Figure 4a shows the bent conformation tetrahydrofolic acid with its Paba residue above the pteridine ring, allowing tight enzyme and nucleotide binding. In this thymidylate synthase-catalyzed reaction, it is possible to visualize the good proximity of cysteine residue( Cys146)sulphydryl group to the C6 of dUMP pyrimidine moiety(1.94 A), and the close contact of the coenzyme that favors the attachment of the dUMP enolate at C5. Hydrogen bonds between Cys 146, Arg 166, and Tyr 94 (Fig 4b)are lso included in the Ts structural domain and probably involved in the bond instability of the enzyme covalent adduct for further cleavage. The students are reminded to look at the potential base Tyr 94 and surrounding water that seem involved in the abstraction of the pyrimidine ring C5 proton of the ternary complex, preceding DHF elimination. In addition, the optimal positioning of C6 THF cofactor and the C5 dUMP exocyclic methylene group for hydride transfer(2. 5 A)and the active site shielding from bulk solvent are influenced by Trp80 and Leul43 residues. Although there are other interactions at the catalytic site, the hydrogen bonds between dUMP and the highly conserved amino acid Asn 177, which encode dump specificity over other nucleotides should be pointed out <www.iupac.org/publications/cd/medicinalchemistry/>
13 Tutorial A Protein Explorer contact analysis of the enzyme-substrate complex, PDB entry 1kzi from E. coli, revealed both the ligand binding place and the binding interactions between substrate and cofactor [9]. Indeed, long-range recognition factors between drug and receptor represented by hydrogen bond may be assessed and inferred in terms of electrostatic character and steric orientation. Figure 4a shows the bent conformation tetrahydrofolic acid with its PABA residue above the pteridine ring, allowing tight enzyme and nucleotide binding. In this thymidylate synthase-catalyzed reaction, it is possible to visualize the good proximity of cysteine residue (Cys146) sulphydryl group to the C6 of dUMP pyrimidine moiety (1.94 Å), and the close contact of the coenzyme, that favors the attachment of the dUMP enolate at C5. Hydrogen bonds between Cys 146, Arg 166, and Tyr 94 (Fig. 4b) are also included in the TS structural domain and probably involved in the bond instability of the enzyme covalent adduct for further cleavage. The students are reminded to look at the potential base Tyr 94 and surrounding water that seem involved in the abstraction of the pyrimidine ring C5 proton of the ternary complex, preceding DHF elimination. In addition, the optimal positioning of C6 THF cofactor and the C5 dUMP exocyclic methylene group for hydride transfer (2.5 Å) and the active site shielding from bulk solvent are influenced by Trp80 and Leu143 residues. Although there are other interactions at the catalytic site, the hydrogen bonds between dUMP and the highly conserved amino acid Asn 177, which encode dUMP specificity over other nucleotides should be pointed out. <www.iupac.org/publications/cd/medicinal_chemistry/> version date: 1 December 2006