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7.

05 Spring 2004 March 19, 2004

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

3. (2000 Exam 2 Question 1, 20 points)


What is the effect of:
(a) (4 pts) enzyme concentration on Km?

(b) (4 pts) enzyme concentration on Vmax?

(c) (4 pts) enzyme concentration on the equilibrium constant?

(d) (4 pts) enzyme concentration on the initial reaction rate?

(e) (4 pts) a non-competitive inhibitor on Km?

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.

5. (2001 Exam 1 Question 1, 15 points)


Consider a human mRNA precursor molecule that is 500 nucleotides long, including its
single 100-nt intron and its 80-nt poly(A) tail. (For this question, disregard its cap.)
(a) (5 pts) How many phosphodiester bonds are there in this pre-mRNA prior to splicing?

(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?

6. (2000 Exam 4 Question 4, 10 points)


Some satellite viruses use self-cleaving ribozymes to process the intermediates of rolling-
circle replication. Given that satellites are under strong evolutionary pressure to have small
genomes, why might they have a selective advantage if they use a ribozyme rather than a
protein enzyme for this job?

Recitation 5 Page 2 of 3
7.05 Spring 2004 March 19, 2004

7. (2001 Exam 4 Question 5, 10 points)


Using the technique of in vitro selection, biochemists are learning that RNA can catalyze
many different types of reactions. Which reaction would be the most important reaction to
demonstrate in order to convince you that the RNA world hypothesis is plausible? Explain
your answer.

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]

(1) The enzyme does not exhibit Michaelis-Menton kinetics.

(2) X is a competitive inhibitor

(3) X is a non-competitive inhibitor

(4) X is an allosteric inhibitor

9. (1996 Exam 1 Question 7, 10 points)


Previously, in was thought that peptidyl trasnferase activity was contained within several of
the proteins in the 50S ribosomal particle. Recently, it was discovered that this dogma is
almost certainly wrong: the peptidyl transferase activity appears to be contained in the 23S
rRNA instead. What is the significance, in terms of evolution, of this new discovery?

Recitation 5 Page 3 of 3
Victor Sai
7.05 Spring 2004
The Central Dogma

DNA

RNA Protein

• Differences between prokaryotes and eukaryotes:


Feature Prokaryotes Eukaryotes
Chromosome shape & Circular, no telomeres Linear, ends extended by
Its replication telomerase
Transcription/ Simultaneous in the cytosol Transcription: in the nucleus;
Translation Translation: in the cytosol
Initiation of Starts at first AUG codon after No Shine-Dalgarno sequence
Translation Shine-Dalgarno sequence needed for initiation
None Cap formation, poly(A)
RNA Processing addition, and splicing
Ribosomes Smaller Larger

• Reverse Transcription in Retroviruses:


1) Reverse Transcription (retroviruses):
Event/Activity Genetic Material
Infection, virus enters cell (+) strand of RNA
RNA-dependent DNA Polymerase activity of RT (−) strand of DNA
RNAse H and DNA-dependent DNA Pol activities of RT (+) strand of DNA
Integration of (+) DNA into host chromosome (+) strand of DNA
2) Reverse Transcriptase:
c DNA or RNA dependent DNA Polymerase, synthesizes in 5’→ 3’ direction
d Degrades RNA
e No proofreading
f Requires primer provided by tRNA
Other RNA viruses use RNA-dependent RNA polymerase and exist either as (+) stranded
RNA viruses or (−) stranded RNA viruses.

• Reverse Transcription in Eukaryotes:


Telomerase uses its own RNA template to make DNA. This prevents shortening of linear
chromosomes during DNA replication. No proofreading is necessary because the
fragment generated by telomerase will not be translated.

Page 1 of 1
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

vo = Initial reaction velocity


Vmax = Maximial velocity
Km = Michaelis constant 0 Km Substrate
[S] = Substrate concentration
Michaelis-Menton Plot
Assumptions made in its derivation:
1) [S] 〉〉 [E] (concentration of substrate is much greater than concentration of enzyme)
2) [ES] is constant (rate of formation of ES equals rate of breakdown of ES)
Conclusions drawn from its derivation:
1) The Michaelis constant, Km, is characteristic of an enzyme and reflects the affinity of
the enzyme for its substrate. It does not vary with the concentration of the enzyme.
2) Km describes the dissociation constant of [ES].
Low Km reflects a high affinity of the enzyme for substrate
High Km reflects a low affinity of the enzyme for substrate
3) Given an enzyme with a particular Km,
V
If [S] 〈〈 Km, then vo = max [ S ] (linear region, 1st order, vo proportional to [S])
Km
If [S] 〉〉 Km, then vo = Vmax (saturated region, 0 zero, vo unaffected by additional [S])
If [S] = Km, then vo = ½ Vmax (velocity reached its half-maximal value)
3. Lineweaver-Burke Plot

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

Page 1 of 2
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

with inhibitor with inhibitor


vo

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

Lineweaver- 1/v o with inhibitor 1/v o with inhibitor


Burke Plot 1/VmaxAPP
w/o inhibitor w/o inhibitor
1/Vmax
1/Vmax

-1/Km -1/Ki 0 1/[S] -1/Km 0 1/[S]

Page 2 of 2
Victor Sai
7.05 Spring 2004
Catalytic RNA

Eight Types of Catalytic RNA (ribozymes):


Types 1-4: Self-cleaving RNAs
Types 5-6: Self-splicing Introns
Type 7: RNAse P
Type 8: Ribosome

Types 1-4: Self-cleaving RNAs


Example 1: Satellite Virus
1) Small circular genome (less than 100 nucleotides)
2) Uses another virus for replication
3) Replicates by a rolling circle mechanism

Rolling circle mechanism:

(-) strand

(+) strand
Small circular genome

Make several copies of its circular


genome, all attached to each other

(-) strand

Cleaves itself

(-) strand

Re-circularizes itself

(-) strand

Repeat entire process

(+) 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.

Hammerhead Enzyme Self-splicing Mechanism:


5' end H
5' end H N H
O

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

Page 2 of 4
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

Group I Intron Splicing Mechanism:

exon 1 Group I Intron exon 2


pUp

Folds around

A
exon 1 Group I Intron exon 2
pUp

Free Guanine from solution G-3'OH Transition State

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

Transition State B exon 1


exon 1 CH 2
from somewhere in the O U
CH 2
O U middle of the intron
A O CH 2 O intron
A O CH 2 O
O OH
H O H intron
Rz-(Mg2+ O O attacks P on O
H
O O intron other exon H O
-
intron O P intron O P O
- OH O
O
Rz-(Mg2+ H
O O Rz-(Mg2+ intron

HO CH 2 O
G
HO CH 2 O
G

Page 3 of 4
Type 7: RNAse P
Trims the 5’end of tRNA

Type 8: Ribosomes
Synthesize proteins by catalyzing formation of the peptide bond during translation.

Page 4 of 4