Biochemistry
Hour exam II
Name:
______________
1. (15 points) List at least 3 different ways to determine the molecular weight of a protein, and give a
brief description of how the method works.
2. (15 points) In lab you are trying to purify a new enzyme. After a simple salt precipitation step you
find your protein contains two contaminants. One contaminant has a molecular weight similar the
enzyme you are trying to purify, but a pI that is two units higher. The second contaminant has a pI that
is almost identical your the enzyme, but has a molecular weight that is four times larger than the
enzyme’s. What further purification steps would you suggest and why?
2
3A (10 points) Below are the v vs. [S] and 1/v vs 1/[S] plots for an enzymatic reaction. How do you
find Vmax and Km in each of these plots.
v (Mol /minute)
Enzymatic Kinetics
140
120
100
80
60
40
20
0
0
0.05
0.1
0.15
[S] (M)
Lineweaver-Burke Plot
0.06
1/v (Min/mole)
0.05
0.04
0.03
Series1
0.02
0.01
0
0
1000
2000
3000
1/[S] (1/M)
B. (5 points) What are the values of Vmax and Km for this enzyme.
C. (5 points) Predict how these two plots would change if we added a competitive inhibitor to the
reaction mix. Show your predicted changes on the above plots.
3
4 A. (10 points) In class we talked about five different general mechanisms that enzymes use to catalyze
reactions. Name and define these 5 general methods:
(You don’t need to fill the page)
4
4B ( 10 points) Below is a mechanism proposed for the enzyme glyceraldehyde-3-phosphate
dehydrogenase
The enzyme catalyzes the reaction :
Glyceraldehyde-3-phosphate + NAD+6Glycerate-1,3-biphosphate + NADH
In this detailed mechanism point out as many places as possible where your general mechanisms from
4A (previous page) are being used.
5
5. (15 points) Give at least two different examples of how covalent modification of protein structure is
used to regulate enzymatic activity. If possible state the name of the enzyme(s) involved, and the names
of any particular residues involved. Are these modifications permanent or reversible?
6 (15 points) Below is are a series of steps thought to be involved in the metabolic pathway. Which
enzymes in this pathway to you think will be regulatory enzymes and why? What substances might be
positive effectors or negative effectors and why?
Enz 5
R
S
N + ATP
Enz 4
Enz 1
O + ADP
Enz 2
P
Enz 3
Q
Enz 6
T
Enz 7
U