ENZYME AND PROTEIN ENGINEERING

UNIT I
1

Enzymes are biological catalysts that speed up chemical reactions by lowering the activation
energy. Imagine a scenario where an enzyme is introduced into a reaction but fails to increase the
reaction rate. Find the potential reasons for this and address them.

Ans:
• Incorrect pH/Temperature
• Low Enzyme Concentration
• Low Substrate Concentration
• Enzyme Inhibitors
• Enzyme Denaturation
• Substrate-Enzyme Mismatch

Match the protein structure levels with their characteristics and functions:
2.
 Ans:
A1-B1-C1
A2-B2-C2
A3-B3-C3
A4-B4-C4

A team of researchers is developing a drug to precisely control the activity of a crucial metabolic enzyme
3. involved in energy production. Guided by the concept of allosteric regulation, they aim to design a
molecule that can either enhance or suppress enzyme activity by binding to a specific allosteric site. Their
goal is to fine-tune metabolic processes to address conditions related to energy imbalances, such as
metabolic disorders or mitochondrial dysfunction. Based on your knowledge and understanding of
allosteric regulation, infer whether the drug should act as an allosteric activator or inhibitor to address
an energy imbalance in cells.

Figure : Allosteric Regulation

Ans:
If the goal is to increase energy production, the drug should act as an allosteric activator.
If the goal is to reduce excess energy production, it should act as an allosteric inhibitor.

Find the group A and group B, given in the figure “Basic structure of Amino acids” which consists
of a central carbon atom, also known as the alpha (α) carbon, bonded to a group ‘A’, group ‘B’ and
4. a hydrogen atom. Group A contains nitrogen, often linked with basic properties, while group B
contains both carbon and oxygen, contributing acidic characteristics.
 Ans:

Group A : Amino Group (-NH2)

Group B: Carboxyl Group (-COOH)

In a biochemical pathway, the enzyme fumarase, a type of lyase, catalyzes the conversion
5. of fumarate to L-malate. A team of researchers is investigating how to regulate fumarase activity
to influence metabolic flux in energy production pathways. By studying the enzyme’s structure
and potential regulatory sites, they aim to develop a molecule that can either enhance or inhibit
fumarase function, providing potential therapeutic strategies for metabolic disorders.Consider a
scenario where a Lactase replaces fumarase in the reaction in the figure “Fumarate’s conversion
to L-malate”, Infer whether the reaction would still proceed efficiently or if an alternative
product might form

Fig.Fumarate’s conversion to L-malate

Ans:
If lactase replaces fumarase, the reaction would not proceed efficiently because lactase
specifically hydrolyzes lactose, not fumarate. An alternative product is unlikely to form, as lactase
lacks the catalytic capability to act on fumarate.

6. Referring to the figure “Feedback Inhibition of Metabolic Pathways,” a team of researchers is

analyzing how substrate movement through a series of enzymes and intermediates is regulated
to maintain metabolic balance. In this pathway, the initial substrate undergoes sequential
transformations, catalyzed by specific enzymes, leading to the formation of an end product.
However, to prevent excessive accumulation, the end product acts as an allosteric inhibitor,
binding to an earlier enzyme in the pathway to reduce its activity. By studying this regulatory
 mechanism, the researchers aim to develop targeted interventions that modulate enzyme
function, ensuring optimal metabolic control in physiological and disease conditions. Emphasis the
movement of substrates and intermediate products through the enzyme and their role of
feedback inhibition in ensuring balanced production. answer shortly

Ans:
• In feedback inhibition, Initialy the substrate moves sequentially through enzymes, forming
intermediate products that lead to the end product.
• When the end product accumulates, it binds allosterically to an earlier enzyme, reducing
its activity.
• This halts excess production, ensuring metabolic balance and efficient resource use.

7. A team of researchers is studying enzyme-substrate interactions to design a drug that can either
enhance or inhibit the activity of a target enzyme involved in a metabolic pathway. The Figure
shows two models of enzyme-substrate binding: A-the lock-and-key model (rigid fit) and the
B- induced-fit model (dynamic conformational change).

Figure: models of enzyme-substrate binding

i) If the researchers want to develop an allosteric drug that precisely modulates the
enzyme's activity, which model lock-and-key or induced-fit would better explain the
drug's mechanism of action? Justify your answer in the context of enzyme regulation
and metabolic control.
ii) In the context of the lock-and-key and induced-fit models shown in the image, how
would an irreversible inhibitor differ from an allosteric inhibitor in its interaction with
the enzyme?
Ans:
i) The induced-fit model better explains the drug's mechanism of action. It accounts for
conformational changes in the enzyme upon binding, which is key for allosteric
regulation, allowing the drug to modulate activity precisely and dynamically, essential
for metabolic control.
ii) An irreversible inhibitor binds covalently to the active site, permanently inactivating
the enzyme, while an allosteric inhibitor binds non-covalently at a separate site,
reversibly altering the enzyme's shape and activity.
8. A research team is studying an enzyme that requires a cofactor for its activity. They observe that
the enzyme is inactive when purified alone but becomes functional when combined with a specific
non-protein molecule.

Figure:Enzyme Components
i) Explain why the purified enzyme is inactive initially and what changes occur to make
it functional. What would this functional complex be called?
ii) In the glucose metabolism. It’s products are Pyruvic acid, ATP and NADH.
Now, due to some malfunction the conversion of NADH to NAD+ is stopped. Give the
impact of this NAD+ reduction in glucose metabolism and discuss about this.
Ans:
i) The purified enzyme is inactive because it lacks its required cofactor (e.g., a
coenzyme), existing as an apoenzyme. When the cofactor binds, it forms the
active holoenzyme complex, enabling catalysis at the catalytic site.
ii) If NAD⁺ is not regenerated, glycolysis halts because NAD⁺ is essential for oxidizing
glyceraldehyde-3-phosphate, leading to decreased ATP production.

(Key terms: Apoenzyme = inactive; Holoenzyme = active with cofactor.)

9. An artificial enzyme is a synthetic organic molecule or ion that recreates one or more functions of
an enzyme. It seeks to deliver catalysis at rates and selectivity observed in naturally occurring
enzymes.
 Figure : Artificial enzymes

1. Predict if we can use artificial enzymes in natural environment just like natural enzymes?
2. Which of the following is not true for synzymes?
a) They are referred to as artificial enzymes
b) They possess two structural entities which are a substrate binding site and a
catalytically effective site
c) They do not obey the Michealis-Menten kinetics
d) Producing catalytic site is difficult
Ans:
1. Artificial enzymes (e.g., nanozymes, semi-artificial enzymes) can function in natural
environments but often with reduced specificity or stability compared to natural
enzymes. Their success depends on design precision and environmental compatibility.
2. Option c) is not true for synzymes. Like natural enzymes, most synzymes obey Michaelis-
Menten kinetics.

(Key points: Artificial enzymes work but may lack natural efficiency; synzymes follow enzyme
kinetics.)

UNIT II
1. 1. Match the following enzymes with their stereospecificity and the type of substrate they
act on:
 Ans:
A1-B1-C1
A2-B2-C2
A3-B3-C3
A4-B4-C4

2. A team of researchers is developing an enzyme matrix entrapment system to enhance enzyme

stability and reusability in biochemical applications.Rearrange the following steps in the correct
order along with the justification for the development of enzyme matrix entrapment:
1)Preparing the enzyme and selecting the appropriate matrix for entrapment.
2)Mixing the enzyme with the matrix and initiating the entrapment process.
3)Optimizing the conditions for the enzyme matrix system (e.g., pH, temperature).
4)Entrapping the enzyme within the matrix, ensuring stability.
5)Testing the effectiveness of the entrapped enzyme in various reactions.
6)Measuring the enzyme activity after entrapment.
a. 1, 2, 3, 4, 5, 6
b. 1, 3, 2, 4, 5, 6
 c. 3, 1, 2, 4, 5, 6
d. 1, 2, 4, 3, 5, 6

Ans: d)1, 2, 4, 3, 5, 6

3. A research team is purifying a mixture of proteins to isolate a target protein essential for a drug
formulation using ion exchange chromatography. They employ a cation exchange column, which
carries negatively charged functional groups. As the protein mixture passes through the column,
proteins with a net positive charge bind strongly to the resin, while negatively or neutrally charged
proteins elute quickly. To achieve optimal separation, the team gradually increases the salt
concentration or adjusts the pH of the elution buffer, selectively displacing bound proteins based
on their charge properties. By fine-tuning these conditions, they aim to isolate the desired protein
with high purity and yield for pharmaceutical applications.

Refer to Figure “The Two Kinds of Ion Exchangers”, and compute the steps to be taken from the
team to recover the protein from the column when the desired protein binds to the cation
exchanger and does not elute with the initial buffer.

Fig:The Two Kinds of Ion Exchangers

Ans:
Steps to Recover the Desired Protein from a Cation Exchange Column:
1. Bind the protein mixture to the cation exchange resin using a low-salt buffer at a pH
where the target protein is positively charged.
2. Wash the column with the same buffer to remove unbound (negatively/neutral)
proteins.
3. Gradually increase salt concentration (e.g., NaCl) or adjust pH to weaken ionic
interactions.
4. Monitor elution using UV absorbance or SDS-PAGE to detect when the target protein
elutes.
5. Collect and pool the fractions containing the purified target protein.

4. Consider you are preparing a dessert that requires milk. The milk contains lactose, which cannot
be directly used as energy by your body. Instead, lactase, an enzyme in your digestive system,
breaks down lactose into simpler sugars that can be absorbed. Based on this scenario, explain the
 reason for lactose is considered a substrate and not an enzyme, while lactase is recognized as an
enzyme.

Ans:
• Lactose is considered a substrate because it is the compound being broken down during
the reaction.
• Lactase is recognized as an enzyme because it catalyzes the breakdown of lactose into
glucose and galactose, making the sugars absorbable by the body. Enzymes like lactase act
on substrates like lactose.

5. In a biochemical pathway, glucose-6-phosphate is converted to glucose by the enzyme glucose-6-

phosphatase, which catalyzes the removal of a phosphate group through a covalent interaction.
This reaction plays a crucial role in maintaining glucose homeostasis, particularly in metabolic
processes like gluconeogenesis and glycogenolysis Consider a scenario where protease replaces
phosphatase in the reaction in the figure “Dephosphorylation of Glucose-6-Phosphate”. Predict
whether the reaction would still proceed efficiently or if an alternative product might form

Figure : Dephosphorylation of Glucose-6-Phosphate

Ans:
If protease replaced glucose-6-phosphatase, the reaction would not proceed efficiently.
Proteases cleave peptide bonds in proteins, not phosphate esters in glucose-6-phosphate.
An alternative product would not form—the substrate would remain unmodified due to enzyme-
substrate mismatch.
Key Reason: Enzymes are highly specific; phosphatases target phosphate groups, while proteases
target proteins.

6. Read the statements and indicate the correct option:

Assertion: Enzyme purity can be assessed by determining the specific activity of the enzyme
preparation.
Reason: Specific activity is the ratio of the amount of enzyme to the total protein content in the
preparation, indicating enzyme efficiency.
A) Both Assertion and Reason are correct, and the Reason correctly explains the Assertion.
B) Both Assertion and Reason are correct, but the Reason does not correctly explain the
Assertion.
C)The Assertion is correct, but the Reason is incorrect.
D) The Assertion is incorrect, but the Reason is correct.

Ans:
A) Both Assertion and Reason are correct, and the Reason correctly explains the Assertion.
7. Milk is a valuable source of nutrients containing protein, fat and the carbohydrate lactose. 5-10%
of the UK population are lactose intolerant. Lactose is a disaccharide that is broken down into
glucose and galactose by the enzyme lactase. (figure)

Figure: Using immobilized enzyme to modify milk.

1. Predict the method of immobilization used in this.

Ans:
Entrapment or Adsorption is likely used for immobilizing lactase in this case.
Reason:
• Entrapment keeps lactase within a gel (e.g., alginate), allowing lactose to diffuse in and
glucose/galactose to diffuse out.
• Adsorption can bind lactase to inert carriers like glass beads, offering simple reuse in
lactose-free milk production.
 UNIT III
1. A typical enzyme inhibitor needs to be analyzed in the context of its mechanism of action and its
effect on reaction kinetics. Do the subjective evaluation and match the given enzyme inhibitors
with their mechanism, affected parameters, and applications.

Ans:

A1-B3-C2-D3
A2-B1-C4-D2
A3-B2-C3-D1
A4-B4-C1-D4

2. You are studying the lactate dehydrogenase (LDH) enzyme, which catalyzes the conversion of
pyruvate to lactate in an ordered sequential mechanism.
Refer to the figure “Reaction of the lactate dehydrogenase: pyruvate (left) is oxidized to lactate
(right) by expense of NADH”. Based on the sequential binding and release process, compute the
order of substrate binding and product release affects the enzyme’s catalytic efficiency.
 Figure : Reaction of the lactate dehydrogenase: pyruvate (left) is oxidized to lactate (right) by
expense of NADH
Ans:
Order of Substrate Binding & Product Release in LDH Reaction:
1. Binding Order:
• NADH binds first (required to activate the enzyme’s catalytic site).
• Pyruvate binds second (substrate positioning for reduction).
2. Catalysis:
• NADH reduces pyruvate → lactate, while NAD⁺ is formed.
3. Release Order:
• Lactate is released first (product of the reaction).
• NAD⁺ is released last (cofactor dissociation completes the cycle).
Effect on Catalytic Efficiency:
✓ Ensures precise alignment for catalysis.
✓ Prevents wasteful binding or incorrect products.
✓ Enhances reaction speed and specificity.

3. A biotechnology company is developing a new industrial process that requires the use of
immobilized enzymes for the synthesis of high-value chemicals. The company is considering
different enzyme immobilization techniques, as shown in the figure titled “Enzyme immobilization
techniques,” to optimize enzyme stability and activity.

Figure - Enzyme immobilization techniques

 Based on the following conditions, predict the most suitable enzyme immobilization method
with justifications for:
a) Enzyme stability under extreme conditions (high temperature or pH)
b) Ease of enzyme separation and reuse
c) Prevention of enzyme leaching during reactions
d) Minimizing diffusion limitations with continuous flow

Ans:
a) Covalent binding
b) Ionic binding
c) Cross-linking
d) Encapsulation

4. You are tasked with optimizing an enzyme’s activity for industrial production. The enzyme shows
peak activity at pH 7.2 to 7.4. However, when the pH rises above 7.5, enzyme activity declines
significantly, and when the pH drops below 7.1, microbial growth becomes uncontrollable.
Additionally, previous experiments have shown that slight fluctuations outside this range have led
to enzyme denaturation or excessive microbial contamination.

Parse a strategy to determine the ideal pH range for maximum enzyme activity and minimal
microbial growth

Ans:
1. Narrow pH Testing: Conduct experiments in small increments (e.g., pH 7.0–7.6) to
pinpoint the exact range where:
• Enzyme activity is ≥90% of peak (likely pH 7.2–7.4).
• Microbial growth is suppressed (pH <7.2 inhibits contaminants).
2. Stability Assays: Incubate the enzyme at candidate pHs (e.g., 7.2, 7.3, 7.4) and measure:
• Activity retention over time (avoid denaturation).
• Microbial counts (ensure pH ≤7.4 prevents contamination).
3. Optimal Compromise: Select the highest pH within 7.2–7.4 that balances:
• Maximal enzyme activity.
• Minimal microbial growth (closer to 7.4 preferred).
4. Buffer System: Use a high-capacity buffer (e.g., HEPES) to maintain pH strictly within ±0.1
units of the chosen value.
Simplify answer:
• Maintain pH between 7.2 and 7.4 for peak enzyme activity.
• Implement buffer systems to prevent fluctuations.
• Regularly monitor contamination risks at lower pH levels.
5. A pharmaceutical company is evaluating a newly developed immobilized enzyme system to
improve enzyme stability and reusability in drug synthesis. Researchers analyze the reaction rates
of free and immobilized enzymes at varying substrate concentrations, as shown in the provided
table titled “Reaction rate comparison: Free versus immobilized enzymes.”
Justify the differences in reaction rates between the two enzyme types, emphasizing the impact
of diffusion limitations on enzyme activity at different substrate concentrations.
Table - Reaction rate comparison: Free versus immobilized enzymes

Substrate Reaction Rate(µmol/min)- Reaction Rate(µmol/min)-

Concentration(mM) Free Enzyme Immobilized Enzyme
2.0 10 7
3.0 20 12
4.0 40 20
20.0 50 25

Ans:
• The free enzyme shows a faster increase in reaction rate with substrate concentration
due to unrestricted substrate access.
• The immobilized enzyme shows lower rates, especially at higher concentrations, due to
diffusion limitations—substrates take longer to reach the enzyme trapped in the matrix.
• This mass transfer resistance reduces the apparent enzyme efficiency at higher substrate
levels. Thus, immobilization limits reaction rate due to slower substrate-enzyme
interaction.
 UNIT IV
1. Select the most appropriate answer after analyzing the following statements:
Assertion (A): Cooperative binding in hemoglobin depends on its quaternary structure.
Reason (R): Conformational shifts responsible for allosteric effects in hemoglobin occur solely
within individual globin chains.
a) Both A and R are true, and R explains A
b) Both A and R are true, but R does not explain A
c) A is true, but R is false
d) A is false, but R is true

Ans:
c) A is true, but R is false
2. You are comparing the heat-induced unfolding behavior of two proteins, Protein X and Protein Y,
using their thermal denaturation profiles shown in the figure titled “Thermal denaturation
profiles.” Protein X shows a gradual loss of structure, with 50% remaining at 60°C and complete
denaturation at 75°C. In contrast, Protein Y maintains 70% of its structure at 60°C but undergoes
a sharp structural loss after 65°C, becoming fully denatured by 85°C.

Figure - Thermal denaturation profiles

Predict the protein with higher potential for successful refolding after heat denaturation. Support
your prediction by analyzing the differences in unfolding transitions and relating them to the
concepts of cooperative versus progressive denaturation behavior.

Ans:
Protein Refolding Potential
Prediction: Protein Y has a higher potential for successful refolding.

• Protein Y exhibits cooperative denaturation,Suggest more structural folading pathway

and greater thermal stability.which is ofter reversible

• Protein X shows progressive denaturation,it Gradual unfolding with increasing

temperature. potentially lower thermal stability., which increase misfolding risks during
refolding.
3. An industrial enzyme has lost its activity due to heat denaturation, and a research lab is
exploring recovery strategies to restore its functional conformation. The experimental teams
apply different interventions known to influence protein folding:
a) Team 1 uses glycerol to stabilize partially unfolded structures
b) Team 2 introduces molecular chaperones to assist in correct refolding
 c) Team 3 combines both glycerol and molecular chaperones in a single recovery system
Identify the strategy likely to yield the highest restoration of enzymatic function and justify the
selection over the other approaches.

Ans:
(c) Team 3 (glycerol + chaperones) is likely to yield the highest restoration of enzymatic function.
Justification:
• Glycerol stabilizes partially unfolded intermediates, preventing aggregation.
• Molecular chaperones actively assist in proper refolding.
Combining glycerol, which stabilizes partially unfolded intermediates, with molecular
chaperones, which assist in correct refolding, provides synergistic support for restoring the
enzyme’s native structure more effectively than either method alone.
4. A pharmaceutical company is investigating the binding efficiency and therapeutic potential of
three ligands—Ligand A, Ligand B, and Ligand C—each targeting the same immune-related cell
surface receptor. Their dissociation constants (Kd) indicate differing binding affinities:
Ligand A: Kd = 10⁻8 M, Ligand B: Kd = 10⁻6 M and Ligand C: Kd = 10⁻4 M.
Each ligand is initially tested at a concentration of 10⁻6 M to assess pharmacodynamic
responses.
a) Identify the ligand expected to maintain receptor occupancy the longest and support your
reasoning.
b) Identify the ligand likely to achieve rapid receptor engagement and provide justification.
c) Identify the ligand expected to show minimal activity when the concentration is decreased to
10⁻4 M and justify the prediction.

Ans:
a) Ligand A – It has the lowest Kd (10⁻⁸ M), indicating highest affinity, so it binds more tightly
and maintains receptor occupancy the longest.
b) Ligand C – It has the highest Kd (10⁻⁴ M), so at 10⁻⁶ M, it's at 100-fold higher than its Kd,
leading to rapid receptor engagement due to excess ligand.
c) Ligand C –it has the highest Kd (10⁻⁴ M) , it show minimal activity and lower concentration
dreastically reduce activity.
5. During a thermal stability experiment, a protein that usually exhibits a slow, progressive rise in
absorbance at 280 nm shows an unexpected abrupt transition at a certain temperature. Evaluate
the underlying factors responsible for this sudden cooperative unfolding event and analyze its
consequences on the integrity of the protein’s tertiary structure.
Ans:
Factors responsible:
• Cooperative unfolding due to disruption of stabilizing interactions (e.g., hydrogen bonds,
hydrophobic core).
• Threshold energy exceeded, triggering simultaneous destabilization of multiple domains.
• Loss of non-covalent interactions maintaining tertiary structure.
• Possible involvement of oligomeric interfaces if the protein is multimeric.
Consequence:
• Abrupt loss of tertiary structure, leading to exposure of hydrophobic regions,
aggregation, and irreversible denaturation.
6. A research team is designing a modular protein composed of four functional domains—Domain
1 (responsible for binding), Domain 2 (catalytic core), Domain 3 (regulatory unit), and Domain 4
(providing structural integrity), as depicted in the figure titled “Protein Architecture”.

Figure - Protein Architecture

They introduce specific modifications to enhance functionality:
a) Oxidation of cysteine residues within the regulatory domain (Domain 3)
b) Strengthening of hydrophobic interactions between the binding and catalytic domains
(Domain 1 and Domain 2) by altering solvent polarity
c) Suppression of glycosylation in the structural domain (Domain 4), alongside exposing the
catalytic domain (Domain 2) to urea
Evaluate the influence of these interventions on inter-domain interactions, the function of each
domain, and the overall activity of the engineered protein.

Ans:
a) Oxidation of cysteine residues in Domain 3:
May form disulfide bonds, potentially altering regulatory control—could enhance or inhibit
domain flexibility and responsiveness.
b) Strengthening hydrophobic interactions (Domains 1 & 2):
Improves domain coupling, likely enhancing substrate binding and catalysis, thus boosting
overall activity.
c) Suppression of glycosylation (Domain 4) + urea exposure (Domain 2):
Leads to reduced structural stability (Domain 4) and partial unfolding/inactivation of catalytic
function (Domain 2), compromising protein integrity and function.

Overall impact: Mixed effects—functional enhancement from (a) and (b), but structural and
catalytic destabilization from (c) may reduce net protein activity.
 UNIT V
1. As the lead investigator in an enzyme modification program, you are tasked with tailoring the
substrate specificity of three key serine proteases—elastase, chymotrypsin, and trypsin—for
targeted catalytic enhancements. The intended modifications aim to enable elastase to act on
basic substrates, optimize chymotrypsin for small hydrophobic molecules, and increase trypsin’s
affinity for aromatic residues without compromising its substrate selectivity.
a) Evaluate the structural and catalytic barriers associated with engineering elastase to
accommodate basic residues in its active site.
b) Assess the enzymatic consequences of altering the volume of chymotrypsin’s substrate-
binding pocket—both in terms of reduction and expansion.
c) Identify the design challenges linked to enhancing trypsin’s interaction with aromatic residues
while maintaining its native specificity profile.

Ans:
a) Elastase modification barriers:
Requires enlarging its narrow, hydrophobic S1 pocket and introducing negatively charged
residues to bind basic substrates—risk of disrupting active site geometry and catalytic
efficiency.
b) Chymotrypsin pocket volume changes:
• Reduction: Impairs binding of bulky substrates, reducing activity.
• Expansion: Decreases specificity, may allow non-specific binding, lowering catalytic
precision.
c) Trypsin design challenges:
Introducing features for aromatic binding (e.g., hydrophobic/aromatic residues) must not
disrupt its Asp189-mediated specificity for basic residues—balancing dual affinity without loss
of selectivity is structurally complex.

2. Evaluate the following statements and choose the most appropriate option:
Statement I: Catalytic antibodies (abzymes) are capable of replicating the enzymatic activity
exhibited by serine proteases through engineered active sites.
Statement II: The catalytic mechanism of abzymes primarily involves stabilization of the
transition state by their antigen-binding (variable) regions.
a) Both statements are correct, and Statement II explains Statement I
b) Both statements are correct, but Statement II does not explain Statement I
c) Statement I is correct, but Statement II is incorrect
d) Statement I is incorrect, but Statement II is correct

Ans: (a) Both statements are correct, and Statement II explains Statement I
3. A pharmaceutical team is developing an antibody-drug conjugate (ADC) using an IgG molecule,
illustrated in the figure titled “IgG structure,” which highlights the Fab, Fc, and hinge regions.
They are assessing suitable sites for attaching the drug to ensure optimal therapeutic function.

Figure - IgG structure

 a) Identify the preferred site (Fab or Fc) for conjugation that avoids disruption of antigen
recognition.
b) Assess the impact of attaching the drug to the hinge region in terms of structural flexibility
and antigen-binding efficiency.

Ans:
a) Preferred site: Fc region – Conjugation here avoids interfering with the Fab region, thus
preserving antigen recognition.
b) Impact of hinge region attachment:
May reduce structural flexibility, potentially hindering Fab movement and antigen-binding
efficiency, especially for targets requiring conformational adaptability.

4. A group of enzymologists is analyzing the activity of native trypsin, modified trypsin, and
elastase on substrates with positive charges, as illustrated in the figure titled “Enzyme
structures.” The modified form of trypsin shows a catalytic efficiency of 120%, which is 40%
higher than the native version (80%), whereas elastase operates at only 40% efficiency.

Figure - Enzyme structures

a) Predict the molecular modifications responsible for the increased catalytic activity of
engineered trypsin compared to its native form.
b) Assess the structural and functional limitations of elastase that contribute to its reduced
efficiency on positively charged substrates.

Ans:
a) Engineered trypsin likely has modifications (e.g., mutations in the substrate-binding pocket or
active site) that enhance substrate affinity or catalytic turnover, increasing efficiency by 40%.
b) Elastase has a narrower binding pocket with bulky residues that hinder access to positively
charged substrates, reducing its catalytic efficiency compared to trypsin.

5. A group of molecular biologists is investigating the catalytic mechanism of serine proteases,

focusing on the functional coordination within the catalytic triad highlighted in the figure titled
“Catalytic triad.” They perform site-directed mutagenesis on individual residues to evaluate their
roles in the catalytic process. A significant drop in catalytic efficiency is noted upon alteration of
specific residues.

Figure - Catalytic triad

 a) Predict which residue—serine, histidine, or aspartate—is primarily responsible for initiating
nucleophilic attack, facilitating proton transfer, and stabilizing the transition state during
catalysis.
b) Assess the consequence of disrupting the internal hydrogen-bonding interactions among the
triad members on the efficiency and integrity of the catalytic cycle.
Ans:
a)
• Serine (Ser195) initiates the nucleophilic attack
• histidine (His57) facilitates proton transfer.
• Aspartate (Asp102) stabilizes the transition state by orienting His57.
b) Disrupting hydrogen bonds in the triad severely impairs catalysis by misaligning residues,
reducing proton transfer efficiency, and destabilizing the transition state.

6. Researchers at a biotechnology institute are evaluating the structural integrity of IgG antibodies,
referring to the figure titled “Structure of IgG antibody.” Their investigation focuses on the
reduction of disulfide linkages that connect the heavy and light chains.

Figure - Structure of IgG antibody

Predict the influence of disrupting these inter-chain disulfide bonds on the antibody’s overall
conformation and assess the resulting changes in antigen-binding affinity.

Ans:
Disrupting the inter-chain disulfide bonds in IgG would:
1. Destabilize the antibody's quaternary structure, leading to misalignment of heavy and
light chains.
2. Reduce antigen-binding affinity due to improper positioning of the Fab regions, critical for
epitope recognition.
Changes in antigen-binding affinity:
The antibody's overall structure would destabilize, causing it to unfold or misfold. This disruption
would affect the antigen-binding sites, leading to a decrease or complete loss of antigen-binding
affinity, as the proper three-dimensional structure required for specific binding would no longer
be maintained.