EXAM 2, BICH 410 (MWF 1:50 PM), Friday, Oct. 20, 1995

Write your name on each page. Write concise answers to demonstrate effectively your mastery of the subject material. Show your work in order to receive partial credit where applicable.

1) (12 pts) Use the information given below to determine the primary structure of a nine amino acid peptide. In order to receive partial credit even if you do not obtain the correct answer or to obtain full credit with the correct answer, you must show your work.
A) The amino acid composition of the peptide is: lysine(2), glycine(2), phenylalanine(2), histidine, leucine, methionine.
B) After incubation of the starting peptide with fluorodinitrobenzene (FDNB), followed by hydrolysis, 2,4-dinitrophenylhistidine was identified by HPLC.
C) When the starting peptide was treated with cyanogen bromide, an octapeptide and free glycine were recovered.
D) When the starting peptide was treated with trypsin, a pentapeptide, a tripeptide and free lysine were obtained. Treatment of the pentapeptide with FDNB gave DNP-histidine. Treatment of the tripeptide with FDNB resulted in DNP-phenylalanine.
E) Digestion of the starting peptide with the enzyme pepsin produced a dipeptide, a tripeptide, and a tetrapeptide. The tetrapeptide was composed of lysine(2), phenylalanine and glycine. The cleavage specificity of pepsin is on the amino terminal side of aromatic amino acid residues.
Write final answer here:
Explain reasoning:

2) (20 pts) Answer the following questions:
A) Name three commonly-used chemical reagents that can denature proteins.
____________________, ___________________, ____________________
B) According to the Bohr effect, a lower ________________ causes a decrease in the affinity of hemoglobin for oxygen.
C) Elastin fibrils are cross-linked by a structure known as _____________________.
D) A modified amino acid prevalent in collagen is _______________________.
E) Silk is composed of protein in ________________________ secondary structure.
It is rich in the following two amino acids: _________________, _________________
F) A conservative amino acid substitution for serine is ________________________.
G) In a right-handed alpha helix, there are ______________ amino acids per turn.
H) Name two ionizable amino acid residues located near the carboxyl terminus of an alpha helix that will stabilize the structure (at pH 7):
____________________, ___________________.

3) (12 pts) An enzyme catalyzes a reaction at a velocity of 20 Ámol/min when the concentration of substrate (S) is 0.01 M. The Km for this substrate is 10-5 M. Assuming that Michaelis-Menten kinetics are followed, what will the reaction velocity be when the concentration of S is:

(a) 10-5 M?
(b) 10-6 M?
For full credit, justify your answers.

4) (13 pts) Consider the oxygen saturation curves shown below and answer the following questions. The curves describe oxygen binding by fetal hemoglobin, adult hemoglobin and myoglobin.
A) What is measured on the Y-axis?
B) Which curve describes oxygen binding by myoglobin? Why?
C) Which curve describes oxygen binding by adult hemoglobin? Why?
D) Which curve describes oxygen binding by fetal hemoglobin? Why? What hemoglobin effector molecule accounts for the difference between fetal and adult hemoglobins?

5) (10 pts) An enzyme follows Michaelis-Menten kinetics. Indicate (with an "X") which of the kinetic parameters in the table would be altered by the following conditions. Give only one answer for each.
(a) 6 M urea
(b) a noncompetitive inhibitor
(c) doubling [S]
(d) a competitive inhibitor
(e) a suicide inhibitor

6) (15 pts) Lineweaver-Burk or double reciprocal plot.
A) Starting with the Michaelis-Menten equation, rearrange it in order to obtain the Lineweaver-Burk equation. Explain why this equation describes a straight line.

B) Draw a typical Lineweaver-Burk (or double reciprocal) plot that describes Michaelis- Menten enzyme kinetics. Label the axes. What is the intercept on each axis?

C) On the same plot above, draw the line to represent addition of a competitive inhibitor.

D) Briefly explain why use of a Lineweaver-Burk plot is better than the initial velocity vs. substrate concentration curve in order to determine kinetic constants for an enzyme.

7) (12 pts) Mechanism of chymotrypsin.
A) Draw the side chains of the catalytic triad and explain the role of each in general acid- base catalysis. Do not write out the entire mechanism to answer this question.

B) Draw the covalent acyl-enzyme intermediate. You need only show the portion of the enzyme and substrate containing covalent bonds (no hydrogen bonding patterns necessary).

8) (6 pts) On the ribbon diagram of the protein shown below, clearly draw and label arrows that identify the following features of protein structure.

(Fig. 7-25(d) from text - "insecticyanin")

A) alpha helix
B) beta sheet
C) beta turn