thymidylate synthase

5LMS 0027: MBIC 5 – Interpretative Assignment The substance Rx437 is a reversible inhibitor of the enzyme thymidylate synthase. This enzyme catalyses the conversion of the nucleotide dUMP to dTMP:

= Rx437

+ N5,N10-methylene-FH4 FH2 +

(dUMP) (FH4 = tetrahydrofolate) (dTMP)

Rx437 was investigated as a potential anti-cancer drug. The effect of Rx437 on the apparent Michaelis-constant KM’ and the maximal initial reaction rate vmax of the thymidylate synthase was studied. The results obtained from Lineweaver-Burk plots of the initial rate data are shown in the table below:

[Rx437] / µM 0.0 1.0 2.0 3.0 5.0

KM’ / mM 2.40 12.0 21.6 31.2 50.4

vmax / µM∙s-1 12.1 13.1 12.2 11.9 12.4

1. Suggest why an inhibitor of tymidylate synthase may have anti-tumor activity. (20% of marks)

2. Identify the mechanism of enzyme inhibition by Rx437, and explain your conclusion. (20% of marks)

3. State the Michaelis constant of this enzyme (in the absence of inhibitor) and the inhibitor dissociation constant including correct units. Include in your answer a brief explanation of how you obtained the constants and any graphs you made. (20% of marks)

4. Explain a) why Rx437 may have fewer side effects than methotrexate and b) why infusion of dUMP reduced the side effects of Rx437 even further. (20% of marks)

5. Rx437 is a fluorescent molecule. Explain briefly a) why the fluorescence intensity of Rx437 increases upon binding to thymidylate synthase, and b) why the difference in fluorescence intensity between bound and unbound Rx437 is even larger in a potassium iodide (KI) solution than in pure water. (20% of marks)

The answers to this assignment cannot be found in the literature. You need to use your knowledge of fluorescence, nitrogen metabolism, enzyme kinetics and data analysis/problem solving skills to complete this assignment.

(Provide your answers in HARDCOPY, typewritten in 12pt font size on ONE side of A4 paper, ONE further sheet of paper may be used for plots, parts of the written answer that exceed ONE page will not be marked)

N

N

OH

OH

O

OH

OH O

O

O

P HH

H

N

N

OH

OH

O

OH

OH O

O

O

P HH

H

CH3