Recitation #5
Contact Information
TA: Victor Sai Recitation: Friday, 3-4pm, 2-132
E-mail: sai@mit.edu Office Hours: Friday, 4-5pm, 2-132
Unit 2 Schedule
Recitation/Exam Date Lectures covered
Recitation #5 Friday, March 19 12, 13, 14
Recitation #6 Tuesday, March 30, 5pm, 2-132 15, 16, 17, 18
Exam 2 Review Wednesday, March 31, 7pm, 10-250 10-18
Exam 2 Friday, April 2, 9:30-11am, Walker 10-18
Recitation Overview
Topic Problems
1. Enzyme Kinetics 1, 2, 3, [8]
2. The Central Dogma 4
3. Splicing & Ribozymes 5, 6, 7, [9]
Problems
1. (1996 Exam 2 Question 1, 10 points)
Assume that a particular enzyme-catalyzed reaction follows Michaelis-Menten kinetics with
a Km for the substrate of 1 x 10-6 M. If the reaction rate is 1 x 10-7 mol per minute at a
substrate concentration of 0.1 M, what would the reaction rates be at substrate concentrations
of 0.01 M, 0.001 M, and 1 x 10-6 M? Please explain how your answer was obtained.
2. (a) If Vmax = 100 µmole/mL•sec and Km = 2 mM, what is the velocity of an enzyme-catalyzed
reaction when [S] = 20 mM?
(b) If the total enzyme concentration for this reaction is 200 µM, what is the numerical value
of kcat?
Recitation 5 Page 1 of 3
7.05 Spring 2004 March 19, 2004
4. Suppose that you isolated an HIV strain from a patient’s blood that contained a reverse
transcriptase that proofread. All else being equal, would you consider this virus more or less
of a danger, as compared to the strain that does not proofread? Explain briefly.
(b) (5 pts) How many phosphodiester bonds are there in mature mRNA?
(c) (5 pts) How many phosphodiester bonds are there in the intron product after this
mRNA is spliced by the spliceosome?
Recitation 5 Page 2 of 3
7.05 Spring 2004 March 19, 2004
Practice Problems
8. (2002 Exam 2 Question 3, 10 points)
The following set of velocity curves is obtained for an enzyme-catalyzed reaction in the
presence of increasing amounts of an inhibitor X. Answer the following true or false
questions and justify your answer.
0 mM [X]
10 mM [X]
V0
20 mM [X]
[S]
Recitation 5 Page 3 of 3
Victor Sai
7.05 Spring 2004
The Central Dogma
DNA
RNA Protein
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Victor Sai
7.05 Spring 2004
Enzyme Kinetics
1. Reaction Model
k1 k2
E+S ES E+P
k-1
S: Substrate, E: Enzyme, ES: Enzyme-Substrate complex, k1, k-1, k2: rate constants
k + k2 k
K m = −1 and K D = −1 (KD: dissociation constant)
k1 k1
2. Michaelis-Menten Equation
Vmax
V [S ] k + k2
vo = max where K m = −1
K m + [S ] k1
vo
Vmax/2
Km 1/v o
1 1
= +
vo Vmax [ S ] Vmax
−1 1/Vmax
Intercept on x axis =
Km
1
Intercept on y axis = -1/Km 0 1/[S]
V max
Lineweaver-Burke Plot
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4. Inhibition of Enzyme Activity
Competitive Noncompetitive
Inhibition Inhibition
Inhibitor binds reversibly to the Inhibitor binds reversibly to a site
same site that the substrate would different from the substrate’s
Definition normally occupy and thereby binding site and thereby lowers the
lowers the affinity of the enzyme amount of functional enzyme
for its substrate
Intuition Enzyme sees less substrate Enzyme doesn’t work as well
Effect on Vmax None (same Vmax w/ more substrate) Lowered (slower turnover rate)
Effect on Km Increased (less affinity) None (same affinity for substrate)
Remedy Add an excess of substrate Add extra enzyme
E+S ES E+P E +S ES E+P
+ +
I I
Chemical I
Equation
S EI + S EIS
EI
APP [I ] APP 1
Km = K m (1 + ) Vmax =
Ki [I ]
1+
Vmax [ S ] Ki
V =
[I ] Vmax [ S ]
K m (1 + ) + [ S ] V =
Ki [I ] [I ]
Michaelis- K m (1 + ) + [ S ](1 + )
Menton Plot Ki Ki
w/o inhibitor w/o inhibitor
Vmax Vmax
vo
Vmax/2 Vmax/2
VmaxAPP/2
0 Km Ki Substrate 0 K =K Substrate
m i
[I ] [I ]
K m (1 + ) 1+
1 Ki 1 1 1 K [I ] 1 Ki
= + = m (1 + ) +
V Vmax [ S ] Vmax V Vmax K i [ S ] Vmax
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Victor Sai
7.05 Spring 2004
Catalytic RNA
(-) strand
(+) strand
Small circular genome
(-) strand
Cleaves itself
(-) strand
Re-circularizes itself
(-) strand
(+) strand
Page 1 of 4
Example 2: Hammerhead Enzyme
● Folds into a special conformation that allows self-cleavage between its own C and A residues
● The identical reaction can occur in normal, non-catalytic RNA, but only at 1 reaction per year.
● The special conformation of the folded RNA lowers the activation energy for this reaction and
speeds the reaction to 1 reaction per minute, representing a rate enhancement of 106.
O N H -
O P O
- N
O P O O CH2
O N
N
O CH2
O N O
O cytosine
H O O
cytosine H P
O OH N -
O O
- N H
O P O Self-cleavage H
H N N
O CH2
O N N
N
H H N
HO CH2
adenine O N
N
H
O OH
O
-
P O
adenine
O OH
O
-
O P O
.
3' end
O
.
3' end
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Types 5-6: Self-splicing introns
Two classes of introns splice themselves out without the help of proteins:
● Group I introns: 1011 rate enhancement, uses Mg2+ at its active site and therefore also
known as a “metaloenzyme.” Mechanism shown below.
● Group II introns: same reaction as spliceosome splicing as learned in Unit I but the RNA is
able to splice itself without the spliceosome by folding into a special conformation
Folds around
A
exon 1 Group I Intron exon 2
pUp
exon 1
B Group I Intron exon 2
pU-3'-OH Gp p
pG
exon 1 exon 2
pUp Gp 3'-OH
Group I Intron
exon 1 exon 1
A CH 2
U
CH 2
U
O O
A O CH 2 O intron
O OH O O H O
stabilized by H
O O O intron
O P O P
- -
O H O H
- -
intron O O intron O O
HO CH 2 O HO CH 2 O
G
HO CH 2 O
G
HO CH 2 O
G
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Type 7: RNAse P
Trims the 5’end of tRNA
Type 8: Ribosomes
Synthesize proteins by catalyzing formation of the peptide bond during translation.
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