Lecture Notes

Thymidylate synthase
Learning objectives:

1. Thymine is synthesised from uracil and later see the importance of this
2. Understand the mechanism of thymidylate synthase
3. How enzyme is regulated
4. How vital enzyme is to thymine production
5. Relate this process to cancer treatment and other diseases/infection processes

LO1. THYMINE IS SYNTHESISED FROM URACIL

Background

 Thymidylate synthase is the enzyme that biosynthesises thymine, which is a precursor for DNA biosynthesis
 Thymine is synthesised from uracil relative
- dUMP (deoxyuridine monophosphate) to dTMP (deoxythymidine monophosphate)
- UMP→UDP→dUDP→dUTP→dUMP→dTMP
- deoxyuridine nucelotides are synthesised soley for the purpose of making deoxythymidine nucleotides
- cells convert dUDP→dUTP→dUMP, which is curious for 2 reasons:
1. requires extra energy compared to simply converting dUDP TO dUMP
2. dUTP can be recognised by DNA polymerase an occasionally result in incorporation of uracil into
DNA in place of thymine
- to minimise this possibility, cells have a lot of the enzymes DUMPase, which catalyse the conversion of dUTP
to dUMP
 U is found in RNA
- There is an exception.
- A few DNA molecules have U instead of thymine
 T is found in DNA
- There is an exception.
- Sometimes transfer RNA molecules contain some T as well as U

dUMP creation

 produced via 2 routes
1. dCMP + H2O → dUMP + NH4+
- Enzyme: dCMP deaminase
2. dUTP + H2O → dUMP + PPi
- enzyme: dUTP diphosphohydrolase
 Why this second route?
- With increased amounts of dUTP = Increased chance of incorporation into DNA
- Enzymes that create DNA cannot easily tell between dUTP & dTTP
- Creation as dUMP lowers concentration of dTTP
- Now more controlled production as a result

Thymidylate synthase

 TS uses a cofactor for addition of methyl group to dUMP
- N5N10-methylene- THF cofactor
 During reaction THF is oxidised to dihydrofolate (DHF)

, Tetrahydrofolate

 In the C1 unit pool there is a resource of C1 carbons at different oxidation states
- Use the different oxidation states to come in to different systems in different processes
- i.e. oxaloacetate from pyruvate via addition of a carboxyl group
- note: biotin acts as the activated carrier of CO 2
- THF can transfer a carbon and also be a carrier in some reactions
More versatile (remember C1 unit pool)

Structure of thymidylate synthase

 265 residues
 Classified as an αβ protein
- Comprises of α helices & β sheets
 TS is a 2-layer sandwitch

LO3. HOW THE ENZYME IS REGULATED

Background of enzyme reaction

 Take the 6’ of the N5N10-methylene- THF cofactor and tritiate it (= make it radioactive)
 Incubate TS with [6-3H] N5N10-methylene- THF and dUMP
- The H on the 6’ becomes part of the methyl group in the thymine monophosphate (dTMP) that is generated
 Incubate TS with [5-3H]dUMP & N5N10-methylene- THF
- Find 3H into H2O
 Uracil C6 position is the β positon of an αβ-unsaturated ketone
- This makes it susceptible to nucleophilic attack
- Need a nucleophile to carry out a nucleophilic attack in the enzyme
Nucleophile is Cys146
Found as a negatively charged side chain thiolate
In general, it is a good idea to have other residues around to stabilise the negatively charged side
chain
In this example, the positively charged Arg166 residue stabilises the negatively charged Cys146 side
chain