Biomedical Engineering: Bridging Medicine and Technology 1st Edition by Veronique Tran,Mark Saltzman

Exercise 2

Penicillin is hydrolyzed and thereby rendered inactive by penicillinase (also known as β-lactamase), an enzyme present in some resistant bacteria. The mass of this enzyme in Staphylococcus aureus is 29.6 kd. The amount of penicillin hydrolyzed in 1 minute in a 10-mL solution containing 10−9 g of purified penicillinase was measured as a function of the concentration of penicillin. Assume that the concentration of penicillin does not change appreciably during the assay. [Note that kilodalton is the unit for MW for biological molecules. A dalton is another name for atomic mass unit. For example, a protein with a mass of 15 kd means that the MW is 15,000 g/mol.]
a. Plot V0 versus [S] for these data. Does penicillinase appear to obey Michaelis-Menten kinetics? If so, what is the value of Km?
b. What is the value of Vmax? Indicate on the plot how you determined Km and Vmax.
c. Write the Michaelis-Menten equation and show the derivation of the Lineweaver-Burke equation.
d. Plot 1/V0 versus 1/[S].
e. Determine Km and Vmax using the Lineweaver-Burke plot. Indicate on
the plot how you determined Km and Vmax.
f. Assume there is one active site per enzyme molecule. What is the
turnover number of penicillinase under these experimental conditions?
g. How much time does it take for the penicillinase to hydrolyze one
penicillin molecule?