7.

016: Fall 2018: MIT

7.016 Recitation 3 – Fall 2018

(Note: The recitation summary should NOT be regarded as the substitute for lectures)
(This material is COPYRIGHT protected.)

Summary of Lecture 4 (9/12):

Enzymes and energy: Enzymes are biological catalysts. Most biological catalysts are proteins,
although RNA can also have enzymatic role and catalytically- active RNAs are known as ribozymes.
Enzymes catalyze specific biological reactions and act by lowering the activation energy of the reaction
that they catalyze. Each enzyme has a specific 3-D conformation and an active site(s) to which the
substrate molecules can bind to form a transition state enzyme-substrate complex (ES complex). The
complex then gives rise to product (P) and the enzyme is released in its original form to catalyze the
reaction once again.

There may be different forms of the same enzymes (isozymes) that have different physical properties
but which catalyze the same reaction.

Each enzyme is specific for a particular reaction. Enzyme function may be regulated by various factors.
These factors may include i.e. prosthetic groups, metal ions/cofactors, coenzyme, substrate
concentration, pH, temperature, inhibitors, competitive or non-competitive inhibitors, allosteric
modulators and the feedback inhibition by the end product of a biochemical reaction that involves
multiple reaction steps.

Reaction kinetics: An endergonic reaction (one with a positive ΔG) cannot occur spontaneously,
although it may be coupled to an exergonic reaction. In some cases exergonic reaction (one with a
negative ΔG) can proceed spontaneously. The thermodynamics of the reaction are dictated by the
difference in free energy between the substrate and the products. The kinetics of the reaction is
determined by the transition stage and how much energy must be added to form the high energy
intermediate. Enzymes lower the activation energy needed for a reaction to proceed, but do not
change the free energy (ΔG) of either the reactants or the products. The following equation relates ΔG
to enthalpy (ΔH, the available energy) and entropy (ΔS, unusable energy).

ΔG = ΔH - TΔS where T is the absolute temperature.

Questions:
1. Leu-Enkephalin is a bioactive pentapeptide (5 amino acids) that is involved in natural pain regulation.
The sequence is: Tyr-Gly-Gly-Phe-Met

a) Draw an accurate chemical structure of Leu-enkephalin using a “line-angle” rendition.

b) Label the following on your structure: the C-terminus, the N-terminus, the aromatic side chains of the
residues, the a-carbons and the peptide bonds.

1 Diviya Ray
 7.016: Fall 2018: MIT

2. Trypsin is a protease. This enzyme breaks a protein into peptides by hydrolyzing the peptide bonds
that have amino acids lysine and arginine at the carboxyl (-COOH) side of the peptide bond. The steps
involved in the production and regulation of trypsin are outlined and also shown in the schematic below.
Please note that a “T” represents inhibition and an “->” represents activation.

• Reaction 1: Trypsin is produced as inactive trypsinogen.

• Reaction 2: Trypsinogen is cleaved to active trypsin and a hexapeptide by the action of enteropeptidase.
• Reaction 3: Trypsin hydrolyzes the peptide bonds that have lysine and arginine at the carboxyl (-COOH)
side of the cleaved peptide bond.
• Reaction 4: Trypsin then undergoes feedback inhibition by cholecystokinin.

cholecystokinin
Protein
Trypsinogen Trypsin
Enteropeptidase
Peptides
Hexapeptide
a) Summarize the effect of enteropeptidase on the reaction parameters specified in the table below.

Parameters Enteropeptidase

Is the reaction catabolic/ anabolic?

What is the effect of enzyme on the reaction equilibrium (Keq) (increases/

decreases/ unchanged)?
What is the effect of enzyme on the enthalpy of reaction (H) (increases/ decreases/
unchanged)?
What is the effect of enzyme on the entropy of reaction (S) (increases/ decreases/
unchanged)?

You mimic Reaction #3 in five separate test tubes (1-5) as described below. You allow the reaction to
proceed for 30 minutes in each tube and measure the amount of protein hydrolyzed.
o
• Tube #1 (Control): You perform the reaction at 37 C and pH 7.4 and measure 100% hydrolysis of the
protein into peptides.
o
• Tube #2: You perform the reaction at 50 C and pH 7.4 and observe 0% hydrolysis of the protein
o
substrate. However, if the temperature is brought to 37 C, you observe 100% hydrolysis of the protein as
seen in tube #1.
o ++
• Tube #3: You perform the reaction at 37 C and pH 7.4 for and in the presence of EGTA, a Ca ion
chelator (i.e. it quenches / removes the Ca2+ ions) and observe 0% hydrolysis of the protein substrate.
++
You add excess of Ca ions to the tube and observe 100% hydrolysis of the protein.
o
• Tube #4: You perform the reaction at 37 C and a pH of 7.4 in the presence of soybean trypsin
inhibitor (SBI) and do not detect any measurable hydrolysis of the protein. You increase the substrate
concentration by 4 fold and observe 100% hydrolysis of the protein.
o
• Tube #5: You perform the reaction at 37 C and a pH of 7.4 in the presence of di-
isopropylfluorophosphate (DFP) and observe 0% hydrolysis of the protein. You increase the substrate
concentration by 4 fold but do not observe a measurable hydrolysis of protein substrate.

2 Diviya Ray
 7.016: Fall 2018: MIT

b) Explain the effect of the changed reaction parameters in the following test tubes on structure and
function of trypsin and its protein substrate.

Reaction parameters Affects Trypsin (Yes/No)? Explain. Affects Trypsin substrate (Yes/
No)? Explain.
o
50 C in tube #2

EGTA (a chemical that

quenches or removes
the calcium) in tube #3

c) Based on the information provided, would you characterize…

i. SBI as a competitive / non- competitive inhibitor / allosteric modulator? Explain why you
selected this option.

ii. DFP as a competitive/ non- competitive inhibitor / allosteric modulator? Choose all possible
options and give an explanation for the option(s) that you selected.

3 Diviya Ray
 7.016: Fall 2018: MIT

Solutions to Questions:
1. Leu-Enkephalin is a bioactive pentapeptide (5 amino acids) that is involved in natural pain regulation.
The sequence is: Tyr-Gly-Gly-Phe-Met
S

sidechain OH A

O O
H H
N N O
H 3N N N
H H
O O O

Side-
chain

a) Draw an accurate chemical structure of Leu-enkephalin using a “line-angle” rendition.

b) Label the following on your structure: the C-terminus, the N-terminus, the aromatic side chains of the
residues, the a-carbons and the peptide bonds.

2. Trypsin is a protease. This enzyme breaks a protein into peptides by hydrolyzing the peptide bonds
that have amino acids lysine and arginine at the carboxyl (-COOH) side of the peptide bond. The steps
involved in the production and regulation of trypsin are outlined and also shown in the schematic below.
Please note that a “T” represents inhibition and an “->” represents activation.

• Reaction 1: Trypsin is produced as inactive trypsinogen.

• Reaction 2: Trypsinogen is cleaved to active trypsin and a hexapeptide by the action of enteropeptidase.
• Reaction 3: Trypsin hydrolyzes the peptide bonds that have lysine and arginine at the carboxyl (-COOH)
side of the cleaved peptide bond.
• Reaction 4: Trypsin then undergoes feedback inhibition by cholecystokinin.

cholecystokinin
Protein
Trypsinogen Trypsin
Enteropeptidase
Peptides
Hexapeptide
a) Summarize the effect of enteropeptidase on the reaction parameters specified in the table below.

Parameters Enteropeptidase

Is the reaction catabolic/ anabolic? Catabolic

What is the effect of enzyme on the reaction equilibrium (Keq) (increases/ Unchanged
decreases/ unchanged)?
What is the effect of enzyme on the enthalpy of reaction (H) (increases/ decreases/ Unchanged
unchanged)?
What is the effect of enzyme on the entropy of reaction (S) (increases/ decreases/ Unchanged
unchanged)?

4 Diviya Ray
 7.016: Fall 2018: MIT

You mimic Reaction #3 in five separate test tubes (1-5) as described below. You allow the reaction to
proceed for 30 minutes in each tube and measure the amount of protein hydrolyzed.
o
• Tube #1 (Control): You perform the reaction at 37 C and pH 7.4 and measure 100% hydrolysis of the
protein into peptides.
o
• Tube #2: You perform the reaction at 50 C and pH 7.4 and observe 0% hydrolysis of the protein
o
substrate. However, if the temperature is brought to 37 C, you observe 100% hydrolysis of the protein as
seen in tube #1.
o ++
• Tube #3: You perform the reaction at 37 C and pH 7.4 for and in the presence of EGTA, a Ca ion
chelator (i.e. it quenches / removes the Ca2+ ions) and observe 0% hydrolysis of the protein substrate.
++
You add excess of Ca ions to the tube and observe 100% hydrolysis of the protein.
o
• Tube #4: You perform the reaction at 37 C and a pH of 7.4 in the presence of soybean trypsin
inhibitor (SBI) and do not detect any measurable hydrolysis of the protein. You increase the substrate
concentration by 4 fold and observe 100% hydrolysis of the protein.
o
• Tube #5: You perform the reaction at 37 C and a pH of 7.4 in the presence of di-
isopropylfluorophosphate (DFP) and observe 0% hydrolysis of the protein. You increase the substrate
concentration by 4 fold but do not observe a measurable hydrolysis of protein substrate.

b) Explain the effect of the changed reaction parameters in the following test tubes on structure and
function of trypsin and its protein substrate.

Reaction parameters Affects Trypsin (Yes/No)? Explain. Affects Trypsin substrate (Yes/
No)? Explain.
o
50 C in tube #2 Yes, it may denature the enzyme i.e. Yes, since the substrate is a protein
disrupt its active 3D- dimensional itself, an alteration in temperature
conformation. may disrupt its 3D folding.
2+
EGTA (a chemical that Ca ions may act as a prosthetic group No. However, if you make the
2+
quenches or removes for trypsin. Their chelation by EGTA assumption that the Ca ions are
the calcium) in tube #3 may prevent trypsin from catalyzing this needed for the proper folding of
reaction. protein substrate then your answer is
“yes”.

c) Based on the information provided, would you characterize…

i. SBI as a competitive / non- competitive inhibitor / allosteric modulator? Explain why you
selected this option.
The effect of the inhibitor may be reversed by increasing the substrate concentration i.e. it competes
with the substrate to bind to the active site of the enzyme. So SBI is a competitive inhibitor.

ii. DFP as a competitive/ non- competitive inhibitor / allosteric modulator? Choose all possible
options and give an explanation for the option(s) that you selected.
The effect of DFP cannot be reversed by increasing the substrate concentration, suggesting that it
binds to a site on an enzyme other than the substrate-binding site. This makes DFP either a
noncompetitive inhibitor or an allosteric inhibitor.

5 Diviya Ray
MIT OpenCourseWare
https://ocw.mit.edu/

7.016 Introductory Biology

Fall 2018

For information about citing these materials or our Terms of Use, visit: https://ocw.mit.edu/terms.