Q9. A ribozyme (catalytic RNA molecule, E) catalyzes the cleavage of a 7-nucleotide RNA (S7) to

produce a 6 nucleotide RNA (P6). The reaction follows the Michaelis-Menten (MM) kinetics as shown

below (8 points, 2 points each):

S7+ ES7.E=> P6 + E

In an experiment starting concentration of S7 is 150uM, E is 10nM, and Km = 250 nM

A. Why is the second reaction considered irreversible?

B. 20 uM of S7 is converted to P6 in the first 10s of the reaction. Determine the reaction rate (in

M/s) in this experiment/nC. Use the result from B and the MM equation to determine the maximum rate of reaction (in M/s)

at the given ribozyme concentration and k2 for the conversion of S7 to P6.

D. A DNA (D7) is a competitive inhibitor of this reaction. Binding between D7 and E is quantified

by:

E + D7 → E.D7 Ki = [E].[D7]/[E.D7]

In the presence of 10uM D7 the reaction was 50% of the maximal rate when the substrate S7

is 1500 nM. Determine the value of Ki.