Enzymology
Chapter 1: Enzymology
b) Enlist different methods used for purification of an enzyme.
- Methods used for purification of enzyme:
1) Precipitation:Precipitation separates the enzyme from other contaminants based on their differential solubility.
2) Chromatography: Different types of chromatography utilize the distinct properties of enzymes to separate and purify them.
3) Ultrafiltration: This method involves passing the enzyme solution through a filter with a specific molecular weight cutoff. Smaller molecules and impurities are removed while larger enzyme molecules are retained.
4) Electrophoresis: Electrophoretic techniques separate enzymes based on their charge or isoelectric point.
5) Dialysis: Dialysis involves placing the enzyme solution in a dialysis bag or tubing and immersing it in a buffer solution. Smaller molecules, salts, and impurities diffuse out of the bag, while the enzyme is retained.
6) Salting out:By adding salts to the enzyme solution, the proteins can be precipitated and separated from other components.
b) Explain with the help of suitable example the construction of an enzyme
purification chart. [5]
a) Explain concept of an allosteric enzymes with suitable example. [7]
- Concept of Allosteric Enzymes :
1) Allosteric enzymes are special types of enzymes that can change their activity when another molecule binds to a specific site on the enzyme.
2) This regulatory site is called the allosteric site, and it is different from the active site where the enzyme binds to its substrate.
3) When a molecule binds to the allosteric site, it can either increase or decrease the enzyme's activity.
4) The molecule that binds to the allosteric site is called an allosteric effector or modulator.
If the allosteric effector enhances the enzyme's activity, it's called positive allosteric modulation.
5) On the other hand, if the allosteric effector inhibits the enzyme's activity, it's called negative allosteric modulation.
6) Allosteric enzymes often have multiple subunits that work together, and the allosteric site is typically found on specific subunits.
7) When an allosteric effector binds to the allosteric site, it causes a change in the enzyme's shape and structure.
8) This change in shape affects the enzyme's ability to bind to its substrate and perform its catalytic function.
9) By regulating the activity of allosteric enzymes, cells can control important biochemical processes and maintain balance in their metabolic pathways.
Example:
One example of an allosteric enzyme is hemoglobin, the protein responsible for carrying oxygen in our blood. Hemoglobin consists of four subunits, and each subunit can bind to an oxygen molecule.
1) Each subunit of hemoglobin has an allosteric site.
2) When one oxygen molecule binds to a subunit's allosteric site, it causes a conformational change in the protein structure.
3) This conformational change increases the affinity of the other subunits for oxygen.
As a result, the binding of oxygen to one subunit enhances the binding of oxygen to the remaining subunits.
4) This positive allosteric modulation allows hemoglobin to efficiently pick up oxygen in the lungs and release it in tissues where it is needed.
5) Similarly, when oxygen is released from one subunit, it induces a conformational change that decreases the affinity of the other subunits for oxygen.
6) This negative allosteric modulation ensures that oxygen is released in tissues with lower oxygen concentrations.
7) The binding of oxygen at the allosteric sites regulates the overall oxygen-binding and -releasing properties of hemoglobin.
8) This allosteric regulation of hemoglobin enables efficient oxygen transport throughout the body, adapting to the varying oxygen needs of different tissues.
Q. Compare & contrast between KNF & MWC
Comparison of KNF and MWC Models for Allosteric Enzymes
| Aspect | KNF Model | MWC Model |
|---|---|---|
| Definition | Sequential model | Symmetry model |
| Subunit Interaction | Sequential subunit interaction | Simultaneous subunit interaction |
| Active and Relaxed States | T and R states | T (tense) and R (relaxed) states |
| Conformational Change | Triggered by ligand binding | Cooperative conformational change |
| Allosteric Site | Separate site for allosteric regulator binding | Common site for ligand and allosteric regulator binding |
| Cooperative Binding | No explicit treatment of cooperativity | Explains cooperativity based on concerted subunit conformational change |
| Ligand Binding Affinity | Independent binding affinity for each subunit | Ligand binding affinity depends on the conformational state of subunits |
| Hill Coefficient | Hill coefficient not explicitly considered | Hill coefficient can be derived from the model |
| Binding Curves | Non-cooperative binding curves | Cooperative binding curves |
| Ligand Saturation Behavior | Hyperbolic saturation behavior | Sigmoidal saturation behavior |
| Assumptions | No explicit quaternary structural change | Quaternary structural changes occur upon ligand binding |
| Examples | Hemoglobin, myoglobin | Hemoglobin, phosphofructokinase |
model.
Derive kinetic equation for MWC model. [7]
Q. Explain KNF model of allosteric enzymes.
- Explanation of the KNF (Koshland, Némethy, and Filmer) model of allosteric enzymes:
1) The KNF model says that allosteric enzymes have two forms: relaxed (R) and tense (T).
2) When a substrate binds to one part of the enzyme, it changes the shape of that part from T to R.
3) This shape change spreads to the other parts of the enzyme, causing cooperative interactions.
4) In the KNF model, each part of the enzyme can be either T or R independently.
5) The transition between T and R happens through equilibrium constants called K1 and K2.
6) When a substrate binds to the R form, it increases the affinity of other parts for the substrate.
7) Inhibitors can bind to the T form and affect the affinity of other parts for the substrate, causing cooperative inhibition.
8) The KNF model doesn't consider major structural changes in the enzyme upon ligand binding.
9) The binding affinity of the enzyme for the substrate can be described by a formula involving substrate concentration (S) and binding constants (K1 and K2).
10) The KNF model helps explain the sigmoidal (S-shaped) curves and cooperative binding of allosteric enzymes, but real enzymes can have more complex mechanisms.
Q. Derive MM equation for competitive inhibition of enzyme.
-
Derivation of the Michaelis-Menten equation for competitive inhibition in simpler language:
1) Start with the basic Michaelis-Menten equation: V0 = (Vmax * [S]) / (Km + [S]), where V0 is the initial velocity of the reaction, Vmax is the maximum velocity, [S] is the substrate concentration, and Km is the Michaelis-Menten constant.
2) In competitive inhibition, an inhibitor molecule competes with the substrate for binding to the enzyme's active site.
3) Assume that the inhibitor and substrate have the same binding site on the enzyme and compete with each other.
4) Let's denote the inhibitor concentration as [I] and the inhibition constant as Ki.
5) The inhibitor binds reversibly to the enzyme, forming an enzyme-inhibitor complex (EI), preventing the formation of the enzyme-substrate complex (ES).
6) The presence of the inhibitor affects the apparent Km value, which represents the substrate concentration at half of the maximum velocity.
7) To account for competitive inhibition, modify the Michaelis-Menten equation: V0 = (Vmax * [S]) / (Km * (1 + ([I] / Ki)) + [S]).
8) In this modified equation, the denominator (Km * (1 + ([I] / Ki)) + [S]) represents the effective substrate concentration in the presence of the inhibitor.
9) As the inhibitor concentration increases, the denominator becomes larger, resulting in a higher apparent Km value.
10) The increased apparent Km indicates reduced affinity between the enzyme and substrate due to the presence of the inhibitor.
Q. Derive MM equation for enzyme catalyzed reaction in presence of competitive inhibitor.
-
b) Draw secondary plots for uncompetitive inhibition & comment on
calculation of Ki. [5]
- b) Draw secondary plots for non competitive inhibition. [5]
c) Short note on Hill plot.
a) Discuss steps involved in king Altman approach to derive any two
substrate enzyme catalysed reaction. [7]
-1) Define the Reaction: Identify the enzyme-catalyzed reaction you want to study, which involves two substrates (A and B) and the formation of a product (P). The reaction can be represented as: A + B ⇌ P.
2) Set Up Initial Reaction Conditions: Prepare a reaction mixture containing the enzyme, substrate A, substrate B, and any other necessary components required for the reaction.
3) Vary Substrate Concentrations: Prepare a series of reaction mixtures with different concentrations of substrate A while keeping the concentration of substrate B constant. Start with a low concentration of substrate A and gradually increase it for each reaction mixture.
4) Measure Initial Reaction Velocity: Measure the initial velocity of the reaction for each substrate A concentration. This can be done by measuring the rate of product formation over a short period of time.
5) Plot the Data: Create a graph with the initial reaction velocity (V0, y-axis) plotted against the concentration of substrate A ([A], x-axis). Each data point represents a different substrate A concentration.
6) Determine the Initial Velocity at Various Substrate B Concentrations: Repeat steps 3-5, but this time keep the concentration of substrate A constant and vary the concentration of substrate B. Measure the initial reaction velocity for each substrate B concentration and plot the data.
7) Analyze the Graphs: By examining the plotted data, you can determine the relationship between substrate concentrations and initial reaction velocity. The relationship can be described by the following equations:
For substrate A:
V0 = (Vmax / (Km + [A])) * [A]
For substrate B:
V0 = (Vmax / (Km' + [B])) * [A']
8) In these equations, V0 represents the initial velocity, Vmax is the maximum velocity of the reaction, Km and Km' are the Michaelis-Menten constants for substrates A and B, [A] is the concentration of substrate A, and [A'] is the concentration of substrate A when substrate B is varied.
9) Calculate Kinetic Parameters: Use mathematical analysis, such as curve fitting or linear regression, to determine the values of Vmax, Km, and Km' from the plotted data. These parameters provide insights into the enzyme-substrate interactions and the efficiency of the catalytic process.
Interpret the Results: Analyze the derived kinetic parameters to understand how the enzyme interacts with the substrates and how it catalyzes the reaction. The Km values indicate the substrate concentrations at which the enzyme works at half of its maximum velocity, and Km' represents the affinity of the enzyme for substrate B in the presence of substrate A.
Chapter 2: Bioenergetics
-c) Define Gibb’s free energy.
-
Q. State laws of thermodynamics and state their role in biochemistry.
- Here are the three laws of thermodynamics and their roles in biochemistry:
1) First Law of Thermodynamics (Law of Energy Conservation):
- The first law states that energy cannot be created or destroyed; it can only be converted from one form to another.
- In biochemistry, this law is essential in understanding energy metabolism. It helps explain how energy is transferred and transformed within living systems, such as during cellular respiration and photosynthesis. It emphasizes the conservation of energy as it is converted from one molecule to another, enabling the maintenance of vital biological processes.
2) Second Law of Thermodynamics (Law of Entropy):
- The second law states that the entropy (degree of disorder) of an isolated system tends to increase over time.
- In biochemistry, this law is significant in understanding the concept of entropy and its role in chemical reactions and biological processes. It helps explain why some reactions and processes are spontaneous (increase in entropy) while others are not.
3) Third Law of Thermodynamics:
-The third law states that the entropy of a perfectly ordered crystal at absolute zero temperature is zero.
-In biochemistry, the third law is not directly applicable to biological systems. However, it provides a reference point for entropy calculations and comparisons.
a) Comment on entropy. [7]
-
Q. Explain concept of free energy
-c) Write short note on Atkinson’s energy charge.
b) Define enthalpy.
-
Enthalpy, often represented by the symbol H, is a thermodynamic property that describes the heat energy content of a system at constant pressure. It is a measure of the internal energy of a system plus the product of pressure and volume.
In simpler terms, enthalpy represents the total heat energy present in a system and includes the heat exchanged between the system and its surroundings.
Enthalpy is defined by the equation:
H = E + PV
where:
H is the enthalpy of the system,
E is the internal energy of the system,
P is the pressure, and
V is the volume.
b) Write a note on Atkinson’s energy charge. [5]
-b) Short note on high energy compounds.
- Explanation of high-energy compounds using simpler language:
1) Definition: High-energy compounds are special molecules that store a lot of energy in their bonds.
2) Example: The most famous high-energy compound is ATP, which is like a "cellular battery" that provides energy for many processes in our bodies.
3) Energy Release: When ATP breaks a specific bond, it releases a burst of energy that cells can use to do work, such as contracting muscles or building new molecules.
4) Recharging: After ATP releases energy, it becomes ADP. But cells can recharge ADP back into ATP, so it can be used again.
5) Other Examples: There are similar high-energy compounds like GTP, which helps with cell signaling, and acetyl-CoA, which is important for energy metabolism.
6) Role in Metabolism: High-energy compounds are involved in different pathways that break down food molecules and convert them into usable energy for our bodies.
7) Storage and Transfer: Cells store high-energy compounds, like ATP, to have a ready supply of energy when needed. Enzymes and other molecules help transfer and use this energy as required.
8) Overall, high-energy compounds are like energy packets that cells use to do their jobs. They are stored, released, and recharged to keep our bodies running smoothly.
Q. Justify succinate COA is high energy compound.
-
1) Succinyl-CoA has a special bond called a thioester bond that stores a lot of energy.
2) This bond forms between two molecules, succinyl and Coenzyme A (CoA).
3) Succinyl-CoA is an important part of a cycle in the cell called the citric acid cycle, which helps generate energy.
4) It plays a key role in producing ATP, which is like the fuel that powers the cell.
Succinyl-CoA helps make ATP directly by transferring a piece called a phosphate to another molecule.
5) This phosphate transfer creates a molecule called GTP, which can be turned into ATP easily.
6) Succinyl-CoA also contributes to making other energy-rich molecules, like NADH and FADH2.
7) These molecules are used to make ATP in a process called oxidative phosphorylation.
8) When succinyl-CoA is broken down in the citric acid cycle, it produces NADH and FADH2, which carry high-energy electrons to help make ATP.
9) Besides making energy, succinyl-CoA is used as a building block to create important molecules like heme, which is needed for things like hemoglobin and certain enzymes in the body.
Q. Justify ATP is high energy compound.
Chapter 3: Lipid Chemistry and Metabolism
e) Write down two examples of saturated fatty acids with structures.
a) Phosphoglucomutase is crucial for glycogen breakdown as well as
glycogen synthesis. Explain the role of this enzyme in each of the two
processes. [7]
-
Role of phosphoglucomutase in glycogen breakdown and glycogen synthesis:
1) Glycogen Breakdown (Glycogenolysis):
-Phosphoglucomutase helps convert a molecule called glucose-1-phosphate into another molecule called glucose-6-phosphate.
-This conversion is important because glucose-1-phosphate cannot be used directly by the cell, but glucose-6-phosphate can.
-By converting glucose-1-phosphate to glucose-6-phosphate, phosphoglucomutase enables the cell to use glucose-6-phosphate for energy production or other cellular processes.
2) Glycogen Synthesis (Glycogenesis):
- Phosphoglucomutase also plays a role in making glycogen by converting glucose-6-phosphate into glucose-1-phosphate.
- Glucose-1-phosphate is a building block needed to create glycogen.
- Phosphoglucomutase helps in this conversion so that glucose-6-phosphate can be used to build glycogen molecules.
Q. Give role of lipids as signal molecules.
-Lipids play important roles as signal molecules in cellular communication and signaling pathways. Here are the key roles of lipids as signal molecules:
1) Second Messengers: Lipids, such as phospholipids and diacylglycerol (DAG), act as second messengers in signal transduction pathways.
2) Cell Signaling Cascades: Lipids, such as prostaglandins and leukotrienes, act as signaling molecules in inflammatory and immune responses.
3) Hormone Regulation: Some lipids function as hormones or hormone-like molecules, regulating various physiological processes.
4) Membrane Fluidity and Receptor Function: Lipids, particularly phospholipids, are major components of cell membranes.
5) Lipid Signaling in the Brain: Lipids, such as neurotransmitters and endocannabinoids, participate in neuronal communication and neurotransmission in the brain.
a) Write short note on phospholipids.
-1) Phospholipids are special types of fats found in cells.
2) They have a unique structure with a head and tail.
3) Phospholipids make up the walls of cells, like a protective layer.4)
4) They help control what goes in and out of cells.
5) Phospholipids also play a role in keeping the shape of cells intact.
6) They can send signals to other cells, acting like messengers.
7) Phospholipids are involved in breaking down and absorbing fats during digestion.
8) They help transport fats in the body through the bloodstream.
9) Phospholipids are important for keeping cells organized and functioning properly.
b) Outline pathway of biosynthesis of sterols.
a) What are unsaturated fatty acids?
-
1) Unsaturated fatty acids are special kinds of fats. They have bends or kinks in their structure because of double bonds. These fats are often found in plant oils and fatty fish.
2) Unsaturated fats are considered healthier than other types of fats. They can help improve heart health by reducing "bad" cholesterol levels and increasing "good" cholesterol levels.
3) Unsaturated fats are also important for keeping our cells working properly. They help make our cell membranes flexible and fluid, which is necessary for normal cell function.
4) You can find unsaturated fats in foods like olive oil, canola oil, avocados, nuts, and seeds. Including these foods in your diet can have positive effects on your health.
5) In summary, unsaturated fatty acids are special fats with bends or kinks. They are found in plant oils and fish, and they help keep our cells healthy and our hearts happy.
a) Describe in detail synthesis of fatty acids. [7]
- 1) Building Blocks: The starting material for making fatty acids is a molecule called acetyl-CoA, which comes from the breakdown of food or other sources.
2) Enzymatic Reactions: The process takes place in the cell's cytoplasm and involves a series of enzyme-driven reactions.
3) Assembly Line: Think of it as an assembly line, where each step adds a building block to create the fatty acid chain.
4) Adding Carbons: Acetyl-CoA combines with another molecule called malonyl-CoA. They join together, and two carbon atoms from the malonyl-CoA are added to the growing fatty acid chain.
5) Lengthening the Chain: The process is repeated several times, with two-carbon units being added each time, which makes the fatty acid chain longer.
6) Finishing Touches: Eventually, the chain reaches the desired length, and the fatty acid is released as a complete molecule.
7) Regulation: The process is carefully regulated by the cell to ensure that fatty acids are produced when needed and in the right amounts.
8) Energy Storage: Fatty acids can be used as a source of energy for the body, or they can be stored as fat for later use.
a) Discuss in detail steps involved in beta oxidation process. [7]
- Steps involved in the beta-oxidation process, which breaks down fatty acids:
1) Activation: The fatty acid combines with a molecule called Coenzyme A (CoA) to form a compound called fatty acyl-CoA.
2) Transport: The fatty acyl-CoA is moved into a part of the cell called the mitochondria, where beta-oxidation occurs.
3) Oxidation: The fatty acyl-CoA undergoes a series of reactions to break off two-carbon units in the form of acetyl-CoA. This step involves the use of enzymes to help with the breakdown.
4) Water Addition: After oxidation, a molecule of water is added to the remaining fatty acyl-CoA.
5) Second Oxidation: The modified molecule from the previous step is oxidized again to produce a compound called ketoacyl-CoA.
6) Splitting: The ketoacyl-CoA is split into an acetyl-CoA molecule and a shorter fatty acyl-CoA chain.
7) Repeat: The process is repeated with the remaining fatty acyl-CoA chain until the entire fatty acid is broken down into acetyl-CoA units.
8) Energy Production: The acetyl-CoA molecules produced during beta-oxidation enter the citric acid cycle, which generates energy in the form of ATP.
9) ATP Generation: Through the citric acid cycle and other processes, ATP is produced, providing energy for various cellular functions.
10) Overall Energy Yield: Beta-oxidation of fatty acids produces ATP, which is the main energy source for cells.
a) Short note on nomen clature of fatty acids.
-
1) Chain Length: Fatty acids can vary in length, typically ranging from 4 to 24 carbon atoms. The number of carbon atoms in the fatty acid chain determines its designation.
2) Saturation: Fatty acids can be classified as saturated or unsaturated based on the presence or absence of double bonds between carbon atoms in the chain. Saturated fatty acids have no double bonds, while unsaturated fatty acids have one or more double bonds.
3) Double Bond Position: If a fatty acid is unsaturated, the position of the double bond(s) is indicated by a numerical value. The numbering starts from the carboxyl group end, with the carbon closest to the double bond being assigned the lowest number.
4) Naming Unsaturated Fatty Acids: Unsaturated fatty acids are named based on the number of carbon atoms in the chain, followed by a colon (:), the number of double bonds, and the position of the first double bond from the carboxyl end. For example, an 18-carbon chain with two double bonds at positions 9 and 12 is called 18:2Δ9,12.
5) Naming Saturated Fatty Acids: Saturated fatty acids are named based on the number of carbon atoms in the chain, followed by the word "acid." For example, a saturated fatty acid with 16 carbon atoms is called palmitic acid.
6) Common Names: Some fatty acids have common names that are widely used. For instance, oleic acid refers to an 18-carbon unsaturated fatty acid with a double bond at position 9.
7) Omega System: Another nomenclature system, known as the omega system, is used to describe unsaturated fatty acids. It indicates the position of the first double bond from the methyl (omega) end of the chain. For example, an omega-3 fatty acid has the first double bond at the third carbon from the methyl end.
Q. Describe types of glycerrophospolipids in energy compounds.
-
Q. Explain steps involved in biosynthesis of triglycerols.
- 1) Starting Materials: To make triglycerides, we need three fatty acids and a molecule called glycerol-3-phosphate.
2) Combining Fatty Acids: The three fatty acids join together with glycerol-3-phosphate to form a molecule called phosphatidic acid.
3) Removing Phosphate: An enzyme removes a phosphate group from phosphatidic acid, resulting in the formation of a molecule called diacylglycerol (DAG).
4) Adding the Third Fatty Acid: Another fatty acid is added to the DAG molecule, resulting in the final triglyceride molecule.
5) Storage: Triglycerides are stored in specialized structures called lipid droplets within cells.
6) Energy Release: When needed, triglycerides can be broken down into fatty acids to provide energy for the body.
Chapter 4: Carbohydrate Chemistry and Metabolism
a) What are sugar epimers?
-Sugar epimers are a type of molecules called stereoisomers. Stereoisomers are having the same atoms but arranged differently in space. So Sugar epimers are different forms of simple sugars.
Sugar epimers are special because they only differ in the way one particular carbon atom is arranged. This carbon atom is important because it helps sugars join together to form bigger molecules. So, sugar epimers are like siblings that have almost the same structure but differ in the arrangement of that one carbon atom.
a) Describe in detail the role of TCA cycle in generating biosynthetic
intermediates. [7]
The TCA cycle is like a power plant inside cells that helps produce energy and building blocks for making important molecules.
1) It starts with a molecule called acetyl-CoA, which comes from the breakdown of food we eat, like carbs and fats.
2) As the TCA cycle progresses, it releases energy in the form of high-energy electrons and hydrogen ions, which are used to make ATP, the cell's energy source.
3) But the TCA cycle doesn't just make energy. It also creates important molecules that cells need for growth and maintenance.
4) These molecules, called biosynthetic intermediates, act as building blocks for making stuff like proteins, DNA, and fats.
5) For example, some intermediates from the TCA cycle are used to make amino acids, the building blocks of proteins.
6) Other intermediates are used to make things like heme, a molecule needed for making hemoglobin, which carries oxygen in our blood.
7) The TCA cycle produces these biosynthetic intermediates by transforming and rearranging the carbon atoms from acetyl-CoA.
8) The cycle is carefully regulated to ensure that enough intermediates are made to meet the cell's needs for growth and repair.
9) Overall, the TCA cycle is like a multitasking powerhouse. It generates energy for the cell while also producing the building blocks needed for making important molecules that keep our bodies functioning properly.
d) Give two examples of sugar acids.
-Two examples of sugar acids are:
Glucuronic Acid: Glucuronic acid is a sugar acid derived from glucose.
Galacturonic Acid: Galacturonic acid is a sugar acid derived from galactose. It is a key component of pectin, a complex carbohydrate found in the cell walls of plants.
f) What are sugar anomers?
- Sugar anomers are two different forms of a sugar molecule that differ in the configuration around the carbon atom known as the anomeric carbon. The anomeric carbon is the carbon atom that is bonded to both an oxygen atom (forming the carbonyl group) and a hydroxyl group. The two forms of sugar anomers are called the α-anomer and the β-anomer.
The conversion between α-anomer and β-anomer is facilitated by a process known as mutarotation. In an aqueous solution, sugars exist in equilibrium between the α-anomer and β-anomer forms, and this equilibrium is influenced by factors such as pH and temperature.
b) Explain the steps involved in glucogeogenesis with structure. [5]
d) Define Kcat.
- Kcat, also known as turnover number or catalytic constant, is a measure of the catalytic efficiency of an enzyme. Kcat is defined as the maximum number of product molecules formed per second by a single enzyme molecule when the enzyme is functioning at its maximum capacity. It is a crucial parameter in characterizing the enzymatic activity and efficiency of an enzyme.
1) Kcat is calculated by dividing the rate of the enzymatic reaction (measured as the number of product molecules formed per unit of time) by the total enzyme concentration. This value represents the turnover rate or catalytic efficiency of the enzyme.
2) A higher Kcat value indicates a more efficient enzyme, as it can convert substrate molecules into products more rapidly. Enzymes with high Kcat values are generally considered to have a strong catalytic activity and are often referred to as "fast" enzymes.
3) Kcat values can vary widely depending on the enzyme and the specific reaction it catalyzes. Different enzymes have evolved to have different Kcat values optimized for their biological functions and specific substrates.
Q. Give significance of kcat, km and catalytic efficiency.
- Significance of Kcat, Km, and catalytic efficiency in enzyme kinetics:
1) Kcat (Turnover Number):
Significance: Kcat represents the maximum number of substrate molecules converted into product per enzyme molecule per unit of time.
Importance:
- Kcat reflects the catalytic activity and speed of the enzyme.
- It indicates how efficiently the enzyme can convert substrate into product.
- Higher Kcat values indicate a faster enzyme with a higher turnover rate.
- Kcat helps compare the catalytic capabilities of different enzymes.
2) Km (Michaelis-Menten Constant):
Significance: Km is a measure of the substrate concentration at which the enzyme operates at half of its maximum velocity.
Importance:
-Km reflects the affinity between the enzyme and its substrate.
-It indicates how easily the enzyme binds to the substrate and forms the enzyme-substrate complex.
-Lower Km values indicate a higher affinity between the enzyme and substrate.
-Km helps assess the efficiency of the enzyme in capturing and processing substrate molecules.
3) Catalytic Efficiency:
Significance: Catalytic efficiency combines both Kcat and Km to evaluate the overall efficiency of the enzyme.
Importance:
-Catalytic efficiency provides a comprehensive measure of enzyme performance.
-It is calculated as the ratio of Kcat to Km.
-Higher catalytic efficiency values indicate a more efficient enzyme.
-Catalytic efficiency allows for comparisons between different enzymes and their effectiveness in converting substrate into product.
b) Calculate G for hydrolysis of ATP at pH7 & 25°C under steady state
conditions (such as might exist in a living cell) in which the
concentrations of ATP, ADP & Pi are maintained at 10–5M, 10–4M &
10–3M respectively. [5]
Q4) Attempt the following :
-a) What are coupled reactions? Discuss the significance of high energy
compounds in such reactions. [7]
-Q.a) What are coupled reactions? Give role of these reactions in metabolism.
Q. Describe sphingo lipis and role in metabolism.
b) A marine microorganism contains an enzymes that hydrolyzes glucose
6 sulfate. The assay is based on rate of glucose formation. The enzyme
is a cell free extract has kinetic constants of Km = 6.7 × 10–4M &
Vmax = 300nmols/lit–1/min–1. Galactose 6 PO4
is a competitive
inhibitor (I). At 10–5 galactose-6-sulphate & 2 × 10–5M glucose-6-
sulfate, was 1.5nmole × lit–1/min–1. Calculate Ki
for galactose-6-
sulfate. [5]
b) What relationship exist in Km & S in enzyme catalysed reaction
proceeds at 75% Vmax. [5]
b) Calculate G' for complete oxidation of lactic acid to CO2
& H2
O given
the information below. How many moles of ATP could be synthesized in
the process at 40% efficiency? [5]
Given
i) Glucose 2 lactic acid
G1 = – 52000 cal / mole
ii) Glucose + 6O2 6CO2
+6H2
O
G2 = – 686,000 cal / mole
Q4) Attempt the following.
a) Discuss steps involved in king Altman approach to derive any two
substrate enzyme catalysed reaction. [7]
b) Calculate G°' values for the reaction given below FADH2
+ 2 Cytochrome
3 C Fe FAD 2H 2e
(Given 1 E 0.18V 0 & 1 E 0.25V 0 F = 23, 063) [5]
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