Assignment Code: BBCCT-107/TMA/2023

Course Code: BBCCT-107

Assignment Name: Enzymes

Year: 2023

Verification Status: Verified by Professor

Attempt all questions.

Q1) Write short note on the following:

a) Non-competitive inhibition

Ans) Non-competitive inhibition is a type of enzyme inhibition where the inhibitor binds to a site on the enzyme that is not the active site, resulting in a change in the enzyme's shape that prevents substrate binding and activity. It is not overcome by increasing substrate concentration.

b) Reaction Rate

Ans) Reaction rate is a measure of how quickly a chemical reaction proceeds over time. It is typically expressed as the change in concentration of a reactant or product per unit of time. Reaction rates are influenced by various factors, such as temperature, concentration, and the presence of catalysts or inhibitors. The rate of a chemical reaction can be determined experimentally by monitoring changes in concentration or other reaction parameters over time. Reaction rates are important in understanding chemical kinetics, which is the study of how chemical reactions occur and how their rates can be influenced or controlled.

c) Co-factor and Co-enzyme

Ans) Co-factors and co-enzymes are molecules that are required by enzymes to carry out their catalytic function.

Co-factors are inorganic molecules, such as metal ions like zinc, iron or magnesium, that are essential for enzyme activity. They often act as electron carriers or participate in catalytic reactions.

Co-enzymes, on the other hand, are organic molecules that function as co-factors. They are usually derived from vitamins and are involved in various metabolic reactions in the body. For example, nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD) are co-enzymes involved in redox reactions.

Both co-factors and co-enzymes play important roles in enzyme activity, allowing enzymes to carry out their specific reactions efficiently. Without them, many enzymes would not be able to function properly.

d) Active site

Ans) The active site is a region on the surface of an enzyme that is responsible for catalysing a specific chemical reaction. It is a highly specific area that is tailored to recognize and bind to a particular substrate molecule. The structure of the active site is determined by the amino acid sequence of the enzyme and the surrounding environment. The active site may contain amino acid residues that interact with the substrate through hydrogen bonding, ionic interactions, or hydrophobic interactions.

The binding of the substrate to the active site induces a conformational change in the enzyme that brings catalytic residues into proximity with the substrate. This can lead to the formation of a transition state complex, which has a higher energy than the substrate or the product. The active site may also contain cofactors, such as metal ions or coenzymes, that are required for catalytic activity. These cofactors can play a key role in stabilizing the transition state complex and promoting the formation of the product.

Q2) (a) Give an overview of enzyme specificity and its different types.

Ans) Enzyme specificity refers to the ability of enzymes to selectively bind and catalyse specific substrates. Enzymes have evolved to recognize and bind to specific molecules based on their chemical and physical properties, such as shape, size, charge, and polarity. Enzyme specificity can be classified into four main types: absolute, relative, group, and stereochemical specificity.

1. Absolute specificity refers to enzymes that can catalyse only one specific substrate. For example, the enzyme urease catalyses the hydrolysis of urea, but cannot catalyse the hydrolysis of any other substrate.

2. Relative specificity refers to enzymes that can catalyse a range of structurally related substrates. For example, the enzyme lactase can catalyse the hydrolysis of lactose, but can also catalyse the hydrolysis of other similar disaccharides.

3. Group specificity refers to enzymes that can catalyse a range of substrates that contain a specific chemical group. For example, the enzyme protease can catalyse the hydrolysis of peptide bonds in a variety of proteins.

4. Stereochemical specificity refers to enzymes that can catalyse a specific reaction at a specific stereoisomeric center. For example, the enzyme lactate dehydrogenase can selectively catalyse the conversion of L-lactate to pyruvate, but not the conversion of D-lactate to pyruvate.
   

Q2) (b) What is the effect of pH on enzyme activity?

Ans) The pH of the surrounding environment can have a significant effect on the activity of enzymes. Enzymes have an optimal pH at which they exhibit the highest activity, and deviations from this optimal pH can decrease enzyme activity or even denature the enzyme. Each enzyme has a specific pH range at which it functions optimally, and this range varies depending on the enzyme's amino acid composition and the nature of the chemical reaction it catalyses. Enzymes that function in acidic environments, such as pepsin in the stomach, have an optimal pH below 7. Enzymes that function in basic environments, such as alkaline phosphatase in the small intestine, have an optimal pH above 7.

When the pH deviates from an enzyme's optimal range, the activity of the enzyme decreases. This is due to changes in the ionization state of amino acid residues in the enzyme's active site, which can affect the enzyme-substrate interaction and the formation of the transition state complex. At extremely high or low pH values, the enzyme may denature, losing its structural integrity and function.

Q3) Explain ordered and random mechanism of bisubstrate enzyme reactions.

Ans) Bisubstrate enzyme reactions are chemical reactions that involve two substrates that bind to a single enzyme in a specific order to form a product. There are two main mechanisms by which bisubstrate enzyme reactions can occur: ordered and random mechanisms.

In the ordered mechanism, the two substrates must bind to the enzyme in a specific order before the reaction can occur. This means that the binding of the first substrate induces a conformational change in the enzyme that allows the second substrate to bind. Once both substrates are bound, the reaction can occur, resulting in the formation of the product.

One example of an enzyme-catalysed reaction that follows an ordered mechanism is the reaction catalysed by the enzyme glyceraldehyde-3-phosphate dehydrogenase. In this reaction, the enzyme first binds the substrate NAD+, followed by the binding of the substrate glyceraldehyde-3-phosphate. The enzyme then catalyses the oxidation of the aldehyde group of glyceraldehyde-3-phosphate, resulting in the formation of the product 1,3-bisphosphoglycerate.

In the random mechanism, the two substrates can bind to the enzyme in any order, and the binding of one substrate does not affect the binding of the other. Once both substrates are bound, the reaction can occur, resulting in the formation of the product.

One example of an enzyme-catalysed reaction that follows a random mechanism is the reaction catalysed by the enzyme glutathione S-transferase. In this reaction, the enzyme can bind either the substrate glutathione or the substrate 1-chloro-2,4-dinitrobenzene first, and the binding of one substrate does not affect the binding of the other. The enzyme then catalyses the transfer of the glutathione to the 1-chloro-2,4-dinitrobenzene, resulting in the formation of the product glutathione-S-1-chloro-2,4-dinitrobenzene.

Q4) Differentiate between:

a) Fischer lock and Key hypothesis and Induced Fit Model

Ans) The Fischer Lock and Key Hypothesis and the Induced Fit Model are two models that describe how enzymes interact with their substrates. While both models describe the interaction between enzymes and substrates, they differ in their assumptions and predictions.

The Fischer Lock and Key Hypothesis was proposed by Emil Fischer in 1894. According to this model, enzymes and substrates fit together like a lock and key. The enzyme has a rigid, complementary active site that fits perfectly with the substrate, like a key fitting into a lock. The shape of the active site is pre-formed and does not change during the reaction. In other words, the substrate must fit precisely into the active site of the enzyme for the reaction to occur. This model assumes that the active site of the enzyme is already in the optimal conformation to catalyse the reaction.

The Induced Fit Model, on the other hand, was proposed by Daniel Koshland in 1958. According to this model, the active site of the enzyme is not rigid and unchanging but can be modified or induced to fit the substrate.

When the substrate binds to the enzyme, the enzyme undergoes a conformational change that causes the active site to become a better fit for the substrate. In other words, the binding of the substrate induces a change in the enzyme's shape to better accommodate the substrate, like a hand fitting into a glove. This model assumes that the active site of the enzyme is not in the optimal conformation until the substrate binds.

b) Hydrolases and Isomerases.

Ans) Hydrolases and isomerases are two types of enzymes that catalyse different types of chemical reactions in biological systems.

Hydrolases are enzymes that catalyse hydrolysis reactions, which involve the cleavage of a chemical bond using water. Hydrolases break down large molecules into smaller molecules by adding a water molecule across a specific bond in the molecule. This results in the separation of the molecule into two smaller components. Examples of hydrolases include lipases, which break down fats into fatty acids and glycerol, and proteases, which break down proteins into amino acids.

Isomerases, on the other hand, are enzymes that catalyse isomerization reactions, which involve the rearrangement of atoms within a molecule to form an isomer. Isomerases can convert a molecule into a different isomer by rearranging the positions of atoms in the molecule without changing the overall chemical formula. Examples of isomerases include aldose-ketose isomerases, which convert aldose sugars to their corresponding ketose sugars, and phosphohexose isomerases, which convert glucose-6-phosphate to fructose-6-phosphate.

Q5) Derive Alberty rate equation for bisubstrate enzyme catalysed reactions.

Ans) The Alberty rate equation is a mathematical equation used to describe the rate of bisubstrate enzyme-catalysed reactions. It was developed by Robert Alberty and describes the rate of a reaction that involves two substrates  and two products . The reaction is catalysed by an enzyme (E) and can proceed through either an ordered or random mechanism.

The Alberty rate equation for an ordered mechanism can be derived as follows:

1) Step 1: Write the overall reaction equation for the bisubstrate enzyme-catalysed reaction:

2) Step 2: Write the rate equation for the formation of the enzyme-substrate complex:

where k1 is the rate constant for the formation of the ES complex.

3) Step 3: Write the rate equation for the formation of the product complex (EP):

where k2 is the rate constant for the breakdown of the EP complex.

4) Step 4: Write the rate equation for the breakdown of the ES complex to form the EP complex:

where k3 is the rate constant for the breakdown of the ES complex.

5) Step 5: Write the overall rate equation for the bisubstrate enzyme-catalysed reaction:

The negative sign indicates that the concentration of the substrate is decreasing with time, while the concentration of the product is increasing.

6) Step 6: Substitute the rate equations for the ES and EP complexes into the overall rate equation:

where ks1 and ks2 are the Michaelis constants for S1 and S2, respectively.

This is the Alberty rate equation for an ordered mechanism. The Alberty rate equation for a random mechanism is similar but involves different rate equations for the formation of the ES and EP complexes.

Part B

Q6) Explain the mechanism of chymotrypsin enzyme.

Ans) Chymotrypsin is a digestive enzyme that is secreted by the pancreas and plays a crucial role in the breakdown of proteins. It is a protease enzyme, which means that it hydrolyses peptide bonds in proteins, breaking them down into smaller peptides and amino acids. The mechanism of chymotrypsin involves several steps:

1. Activation: Chymotrypsin is secreted by the pancreas in an inactive form called chymotrypsinogen. When chymotrypsinogen enters the small intestine, it is activated by another pancreatic enzyme called trypsin, which cleaves a specific peptide bond and converts it to chymotrypsin.

2. Substrate Binding: Once activated, chymotrypsin can bind to its substrate, which is usually a protein. Chymotrypsin has a specific binding pocket that recognizes and binds to certain amino acid sequences, including those that contain aromatic amino acids such as phenylalanine, tryptophan, and tyrosine.

3. Catalysis: Once the substrate is bound, chymotrypsin catalyses the hydrolysis of peptide bonds. Specifically, it cleaves peptide bonds on the carboxyl side of aromatic amino acids, using a serine residue in its active site to attack the carbonyl carbon of the peptide bond.

4. Release of Products: After the peptide bond is cleaved, the resulting products are released. Chymotrypsin can continue to hydrolyse peptide bonds until the entire protein is broken down into smaller peptides and individual amino acids.

Overall, the mechanism of chymotrypsin involves substrate binding, catalysis, and release of products, all of which are mediated by the specific arrangement of amino acid residues in the enzyme's active site.

Q7) Write short note on the following:

a) Biotin

Ans) Biotin, also known as vitamin H, is a water-soluble B-complex vitamin that plays a crucial role in several metabolic pathways in the body. It is a coenzyme that is required for the metabolism of carbohydrates, fats, and proteins, as well as for the synthesis of fatty acids and glucose. One of the most important functions of biotin is to act as a cofactor for carboxylase enzymes. These enzymes catalyse the addition of a carboxyl group to a substrate molecule, a process that is essential for the synthesis of fatty acids, gluconeogenesis, and other metabolic pathways. Biotin helps to activate these enzymes by binding to a specific site on the enzyme and providing a reactive site for carbon dioxide to bind.

b) Tetrahydrofolate

Ans) Tetrahydrofolate is a coenzyme that plays a crucial role in several metabolic processes, including the synthesis of nucleotides and amino acids. It is a derivative of folic acid, a B-vitamin that is found in many foods and is essential for the formation of red blood cells, DNA synthesis, and cell division.

THF acts as a carrier of one-carbon units, which are used in the synthesis of nucleotides, the building blocks of DNA and RNA. It is also required for the conversion of the amino acid homocysteine to methionine, a process that is important for maintaining optimal levels of homocysteine in the blood. High levels of homocysteine are associated with an increased risk of cardiovascular disease.

THF is synthesized in the body from dietary folic acid, which is converted to dihydrofolate and then to THF by the enzyme dihydrofolate reductase. THF can then be converted to other derivatives that are involved in specific metabolic pathways, such as 5,10-methylenetetrahydrofolate and 5-methyltetrahydrofolate.

c) Diagnostic Enzymes

Ans) Diagnostic enzymes are specific enzymes that are used to diagnose and monitor certain medical conditions. They are often measured in blood or other body fluids, and their levels can provide information about the underlying disease or condition. Some examples of diagnostic enzymes include:

1. Creatine Kinase (CK): CK is an enzyme that is found in muscles, and its levels can be measured in the blood to diagnose and monitor conditions such as heart attacks, muscle disorders, and certain types of cancer.

2. Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST): These enzymes are found in the liver, and their levels can be used to diagnose and monitor liver diseases such as hepatitis and cirrhosis.

3. Amylase and Lipase: These enzymes are involved in the digestion of carbohydrates and fats, respectively, and their levels can be measured in the blood to diagnose and monitor conditions such as pancreatitis and other digestive disorders.

4. Alkaline Phosphatase (ALP): ALP is an enzyme that is found in several tissues in the body, including the liver, bones, and intestines. Its levels can be used to diagnose and monitor conditions such as liver disease and bone disorders.

d) Enzymes in baking industry.

Ans) Enzymes play an important role in the baking industry by improving the quality, texture, and shelf-life of baked goods. Enzymes are naturally occurring proteins that catalyse chemical reactions, and they can be used in the baking industry to improve dough handling properties, increase bread volume, and extend shelf-life. Some examples of enzymes used in the baking industry include:

1. Alpha-Amylase: This enzyme breaks down starch into simple sugars, which are then available for yeast fermentation. Alpha-amylase is used to improve dough handling properties and increase bread volume.

2. Protease: This enzyme breaks down proteins in flour, which can improve the texture and crumb of baked goods. Protease is also used to extend the shelf-life of baked goods by breaking down proteins that can cause staling.

3. Xylanase: This enzyme breaks down hemicellulose, a complex carbohydrate found in flour. Xylanase can improve dough handling properties, increase bread volume, and improve the texture and crumb of baked goods.

4. Lipase: This enzyme breaks down fats into fatty acids and glycerol, which can improve the texture and flavour of baked goods. Lipase is also used to extend the shelf-life of baked goods by breaking down fats that can cause rancidity.

Q8) Discuss the Pyruvate Dehydrogenase complex and its mechanism of regulation.

Ans) The Pyruvate Dehydrogenase complex is a large, multi-enzyme complex that catalyses the conversion of pyruvate to acetyl-CoA, which is an important step in cellular respiration. The PDH complex is composed of three enzymes: pyruvate dehydrogenase, dihydrolipoamide acetyltransferase, and dihydrolipoamide dehydrogenase. The mechanism of the PDH complex involves several steps:

1. Pyruvate is transported into the mitochondria where it is converted to acetyl-CoA by the PDH complex.

2. In the first step, pyruvate is oxidized to acetyl-CoA by pyruvate dehydrogenase (E1), which generates NADH and carbon dioxide.

3. The acetyl group is then transferred to dihydrolipoamide, a coenzyme attached to the dihydrolipoamide acetyltransferase (E2) subunit of the complex.

4. The acetyl group is then transferred to CoA, which produces acetyl-CoA and regenerates the dihydrolipoamide coenzyme.

5. Finally, the dihydrolipoamide is oxidized by dihydrolipoamide dehydrogenase (E3), which generates NADH and regenerates the dihydrolipoamide coenzyme.

The activity of the PDH complex is regulated by several factors, including the availability of substrates and the phosphorylation status of the enzymes. Phosphorylation of the E1 subunit by pyruvate dehydrogenase kinase (PDK) inhibits the activity of the PDH complex, while dephosphorylation by pyruvate dehydrogenase phosphatase (PDP) activates the complex.

The regulation of PDH complex activity is important for maintaining energy homeostasis in the cell. When energy is needed, the PDH complex is activated, which allows pyruvate to be converted to acetyl-CoA and enter the citric acid cycle to generate ATP. When energy is abundant, the PDH complex is inhibited, which prevents the further production of ATP and conserves energy resources.

Q9) Differentiate between the following:

i) Covalent and Ionic method of enzyme immobilization

Ans) Enzyme immobilization is a process that involves attaching enzymes to a support matrix, which allows the enzymes to be reused for multiple reactions. There are several methods of enzyme immobilization, including covalent and ionic methods.

The main differences between the covalent and ionic method of enzyme immobilization are as follows:

1. Chemical Bonds: Covalent immobilization involves the formation of strong covalent bonds between the enzyme and the support matrix, while ionic immobilization involves the formation of weaker ionic bonds between the enzyme and the support matrix.

2. Stability: Covalent immobilization produces a more stable bond between the enzyme and the support matrix, which results in higher enzyme activity and stability. Ionic immobilization, on the other hand, produces a less stable bond, which may result in enzyme leakage and decreased activity.

3. pH and Temperature: Covalent immobilization is more resistant to changes in pH and temperature, while ionic immobilization is more sensitive to changes in pH and temperature.

4. Reusability: Both covalent and ionic immobilization allow enzymes to be reused for multiple reactions. However, covalent immobilization is more effective for long-term use due to its higher stability.

ii) Concerted and Cooperative feedback inhibition

Ans) Feedback inhibition is a regulatory mechanism in which the product of a metabolic pathway inhibits an earlier enzyme in the same pathway, thereby reducing the rate of the pathway. There are two types of feedback inhibition: concerted and cooperative.

The main differences between the concerted and cooperative feedback inhibition are as follows:

1. Mechanism of Inhibition: In concerted feedback inhibition, the inhibitor molecule binds to the enzyme and causes a conformational change in the enzyme that inhibits its activity. In cooperative feedback inhibition, the inhibitor molecule binds to a regulatory site on the enzyme and causes a conformational change that affects the activity of other subunits of the enzyme.

2. Hill Coefficient: The Hill coefficient is a measure of cooperativity in enzyme-substrate binding. In concerted feedback inhibition, the Hill coefficient is 1, which indicates that there is no cooperativity. In cooperative feedback inhibition, the Hill coefficient is greater than 1, which indicates positive cooperativity.

3. Enzyme Structure: Concerted feedback inhibition is observed in enzymes that have a single active site and no regulatory subunits, while cooperative feedback inhibition is observed in enzymes that have multiple subunits and regulatory sites.

4. Enzyme Response to Inhibition: In concerted feedback inhibition, the enzyme is completely inhibited when the inhibitor is bound to the enzyme. In cooperative feedback inhibition, the enzyme activity is gradually reduced as more inhibitor molecules bind to the regulatory sites on the enzyme.

Q10) Draw a table specifying five marker enzymes, their organelle site and associated metabolic pathway.

Ans) Living states are in a steady state that is always changing. Over time, the metabolic concentrations don't change. Most of the reactions in a living body that are sped up by enzymes can be turned around. The metabolic pathways are made up of enzyme reactions that are linked by the substrate and the product.

The reactions of enzymes are tightly controlled to make sure that only the necessary reactions happen in the cell and that the extra ones are stopped by inhibition. Biomolecules like carbohydrates, proteins, and fatty acids are made and broken down in ways that depend on each other. Compartmentation makes sure that organelles or their subcellular compartments are in specific places for catabolism or anabolism.