1. For a Michaelis-Menten hydrolase, k1 = 6.2 x 108 M -1 s -1 , k-1 = 3.1 x 105 s -1 , and k2 = 6.9 x 102 s -1 .
a. Calculate Ks and Km for this enzyme. Does this enzyme follow rapid equilibrium kinetics or simply steady state? Justify your answer.
b. What is kcat for this enzyme? Justify your answer.
c. What is the efficiency of this enzyme? Would this enzyme be considered a perfect enzyme? Justify your answer.
d. In a 100µl reaction containing 50 pmol of the enzyme and saturating levels of substrate, what would be the Vmax value expressed in units of M s-1 . Assume 2 active sites per enzyme molecule.
e. To what concentration must the substrate be reduced in order to achieve half this velocity? Justify your answer mathematically.
f. To what must the enzyme concentration be reduced to achieve half the velocity in subpart d? Justify your answer mathematically.
g. Assume you set up a reaction with the hydrolase in buffer containing 3.0 mM substrate and want to add enough of a competitive inhibitor with Ki = 2.2 x 10-5 M to achieve a quarter of maximal velocity. What concentration of the inhibitor must be present in the reaction?
1. For a Michaelis-Menten hydrolase, k1 = 6.2 x 108 M -1 s -1 , k-1 = 3.1 x 105 s -1 , and k2 = 6.9 x 102 s -1 .
a. Calculate Ks and Km for this enzyme. Does this enzyme follow rapid equilibrium kinetics or simply steady state? Justify your answer.
b. What is kcat for this enzyme? Justify your answer.
c. What is the efficiency of this enzyme? Would this enzyme be considered a perfect enzyme? Justify your answer.
d. In a 100µl reaction containing 50 pmol of the enzyme and saturating levels of substrate, what would be the Vmax value expressed in units of M s-1 . Assume 2 active sites per enzyme molecule.
e. To what concentration must the substrate be reduced in order to achieve half this velocity? Justify your answer mathematically.
f. To what must the enzyme concentration be reduced to achieve half the velocity in subpart d? Justify your answer mathematically.
g. Assume you set up a reaction with the hydrolase in buffer containing 3.0 mM substrate and want to add enough of a competitive inhibitor with Ki = 2.2 x 10-5 M to achieve a quarter of maximal velocity. What concentration of the inhibitor must be present in the reaction?
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