MCB 354 - Exam II - Spring 2008

# MCB 354 - Exam II - Spring 2008 - NAME Answer the questions...

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NAME ______________________________________ 1 Answer the questions in the space provided. Please write legibly and show ALL work and calculations. 1. (10 points) The turnover number for an enzyme is known to be 5,000 min -1 . From the following set of data, calculate the K m and the total amount of enzyme present in these experiments. Substrate concentration (mM) Initial velocity ( μ mol/min) 1 167 2 250 4 334 6 376 100 498 1,000 499 (a) K m = __________ (b) Total enzyme = __________ μ mol Ans: K m = about 2 mM (the concentration of S needed to achieve one-half of V max , which is about 500). The total enzyme present is producing about 500 μ mol of product per minute. Because the turnover number is 5,000/min, the amount of enzyme present must be 0.1 μ mol; 1 μ mol of enzyme would produce 5,000 μ mol product/min. 2. (10 points) When 10 μ g of an enzyme of Molecular Weight 50,000 is added to a solution containing its substrate at a concentration one hundred times the K m , it catalyzes the conversion of 75 μ mol of substrate into product in 3 minutes. What is the enzyme's turnover number? Ans: Because the velocity measured occurs far above K m , it represents V max . Ten μ g of the enzyme represents 10 × 10 -6 g/(5 × 10 4 g/mol), or 2 × 10 -10 mol of enzyme. In 3 minutes, this amount of enzyme produced 75 μ mol of product, equivalent to 25 × 10 -6 mol of product per minute. The turnover number is therefore (25 × 10 -6 mol/min)/(2 × 10 -10 mol) = 12.5 × 10 4 min -1 .

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NAME ______________________________________ 2 3. (10 points) The enzymatic activity of lysozyme is optimal at pH 5.2 and decreases above and below this pH value. Lysozyme contains two amino acid residues in the active site essential for catalysis: Glu 35 and Asp 52 . The p K value for the carboxyl side chains of these two residues are 5.9 and 4.5, respectively. What is the ionization state of each residue at the pH optimum of lysozyme? How can the ionization states of these two amino acid residues explain the pH-activity profile of lysozyme? Ans: For the enzyme to be active, it is likely that Asp 52 is unprotonated and Glu 35 is protonated. When the pH is below 4.5, Asp 52 becomes protonated, and when it is above 5.9, Glu 35 is deprotonated, either of which decreases the activity of the enzyme. (See Fig. 6-20, p. 215.)
NAME ______________________________________ 3 4. (20 points)

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MCB 354 - Exam II - Spring 2008 - NAME Answer the questions...

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