Penicillin is hydrolyzed and thereby rendered inactive by penicillinase, an enzyme present in some resistant bacteria. The mass of this enzyme in Staphylococcus aureus is 29.6 kDa. 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 atom
Penicillin is hydrolyzed and thereby rendered inactive by penicillinase, an enzyme present in some resistant bacteria. The mass of this enzyme in Staphylococcus aureus is 29.6 kDa. 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
Penicillin (M) Amount hydrolyzed (nanomoles)
1 0.11
3 0.25
5 0.34
10 0.45
30 0.58
50 0.61
1. Plot Vo versus [S] for these data. Does penicillinase appear to obey Michaelis-Menten kinetics? If so, what is the value of Km ?
2. What is the value of Vmax? Indicate on the plot how you determined Km and
Vmax.
3. Write the Michaelis-Menten equation and show the derivation of the Lineweaver-Burke equation.
4. Plot 1/Vo versus 1/[S].
5. Determine Km and Vmax using the Lineweaver-Burke Plot. Indicate on the plot how you determined Km and Vmax.
6. Assume there is one active site per enzyme molecule, what is the turnover
number of penicillinase under these experimental conditions?
7. How much time does it take for the penicillinase to hydrolyze one penicillin molecule?
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