Dr.Anant Achary and Dr.S.Karthikumar
Department of Biotechnology
Kamaraj College of Engineering and Technology
S.P.G.C.Nagar, Near Virudhunagar, Tamilnadu
INDIA
The document discusses enzyme kinetics models including the Michaelis-Menten model and Lineweaver-Burk double reciprocal plot. The Michaelis-Menten model relates reaction rate to substrate concentration using kinetic constants Km and Vmax. It describes the enzyme-substrate reaction mechanism. The Lineweaver-Burk plot is a graphical representation that transforms the Michaelis-Menten equation into a straight line to determine Km and Vmax. It can distinguish competitive and noncompetitive enzyme inhibition patterns.
The document summarizes the mechanisms of enzyme catalysis. It discusses how enzymes lower the activation energy of reactions by releasing binding energy when interacting with substrates. This allows enzymes to accelerate reactions by stabilizing transition states. There are three main types of catalytic mechanisms: acid-base catalysis, covalent catalysis, and metal ion catalysis. Acid-base catalysis involves proton transfers. Covalent catalysis forms temporary covalent bonds between enzymes and substrates. Metal ion catalysis uses metal ions like iron and copper to orient and stabilize reactive molecules in enzyme active sites.
This document provides an overview of enzyme kinetics concepts including:
1) Rate constants like k1 and k-1 describe the rates of individual reaction steps. The overall rate v depends on reactant concentrations according to rate laws.
2) Michaelis-Menten kinetics describe enzyme-catalyzed reactions using parameters like Km, Vmax, and kcat/Km. Km represents substrate binding affinity, Vmax is the maximum reaction rate, and kcat/Km is the catalytic efficiency.
3) Reversible inhibitors are classified as competitive, non-competitive, or uncompetitive depending on whether they bind the enzyme (E), enzyme-substrate complex (ES), or both.
Fast protein liquid chromatography (FPLC) is a type of liquid chromatography used to analyze or purify proteins. It introduces samples onto a column containing resin beads, then uses buffers to differentially elute bound protein. FPLC allows separation of heat-labile biomolecules like proteins under mild conditions like 4°C. It has advantages like simple reproducible separation, efficient resolution, and support for a wide range of columns and procedures under low pressure. Limitations include needing glass columns and inability to handle high pressures.
Analytical centrifugation is a technique used to characterize macromolecules based on how they sediment in a centrifugal field. The document discusses the instrumentation, working principle, and two main types of analysis - sedimentation velocity and sedimentation equilibrium. Sedimentation velocity provides information about shape, mass, and size by monitoring the boundary formed over time as particles sediment. Sedimentation equilibrium determines mass composition by analyzing the particle distribution once equilibrium between sedimentation and diffusion is reached. Analytical centrifugation is useful for determining properties like molecular weight, stoichiometry, assembly, and conformation.
The document discusses cellular growth modeling and classification, describing various types of models from unstructured and unsegregated to structured and segregated. It also covers the components of model construction including state variables, parameters, equations, and the definition of volumetric and specific rates for microbial growth, death, product formation, and substrate uptake. The classification aims to account for heterogeneity at both the population and intracellular levels in modeling biological systems.
The B cell receptor is a transmembrane protein on B cells that is composed of a membrane-bound immunoglobulin molecule and a signal transduction moiety. The B cell receptor consists of an Ig molecule anchored to the cell's surface and has two key functions: signal transduction upon antigen interaction and internalization of antigens for processing and presentation to T cells. The B cell co-receptor is a complex of CD19, CD21, and CD81 expressed on mature B cells.
The document discusses enzyme kinetics models including the Michaelis-Menten model and Lineweaver-Burk double reciprocal plot. The Michaelis-Menten model relates reaction rate to substrate concentration using kinetic constants Km and Vmax. It describes the enzyme-substrate reaction mechanism. The Lineweaver-Burk plot is a graphical representation that transforms the Michaelis-Menten equation into a straight line to determine Km and Vmax. It can distinguish competitive and noncompetitive enzyme inhibition patterns.
The document summarizes the mechanisms of enzyme catalysis. It discusses how enzymes lower the activation energy of reactions by releasing binding energy when interacting with substrates. This allows enzymes to accelerate reactions by stabilizing transition states. There are three main types of catalytic mechanisms: acid-base catalysis, covalent catalysis, and metal ion catalysis. Acid-base catalysis involves proton transfers. Covalent catalysis forms temporary covalent bonds between enzymes and substrates. Metal ion catalysis uses metal ions like iron and copper to orient and stabilize reactive molecules in enzyme active sites.
This document provides an overview of enzyme kinetics concepts including:
1) Rate constants like k1 and k-1 describe the rates of individual reaction steps. The overall rate v depends on reactant concentrations according to rate laws.
2) Michaelis-Menten kinetics describe enzyme-catalyzed reactions using parameters like Km, Vmax, and kcat/Km. Km represents substrate binding affinity, Vmax is the maximum reaction rate, and kcat/Km is the catalytic efficiency.
3) Reversible inhibitors are classified as competitive, non-competitive, or uncompetitive depending on whether they bind the enzyme (E), enzyme-substrate complex (ES), or both.
Fast protein liquid chromatography (FPLC) is a type of liquid chromatography used to analyze or purify proteins. It introduces samples onto a column containing resin beads, then uses buffers to differentially elute bound protein. FPLC allows separation of heat-labile biomolecules like proteins under mild conditions like 4°C. It has advantages like simple reproducible separation, efficient resolution, and support for a wide range of columns and procedures under low pressure. Limitations include needing glass columns and inability to handle high pressures.
Analytical centrifugation is a technique used to characterize macromolecules based on how they sediment in a centrifugal field. The document discusses the instrumentation, working principle, and two main types of analysis - sedimentation velocity and sedimentation equilibrium. Sedimentation velocity provides information about shape, mass, and size by monitoring the boundary formed over time as particles sediment. Sedimentation equilibrium determines mass composition by analyzing the particle distribution once equilibrium between sedimentation and diffusion is reached. Analytical centrifugation is useful for determining properties like molecular weight, stoichiometry, assembly, and conformation.
The document discusses cellular growth modeling and classification, describing various types of models from unstructured and unsegregated to structured and segregated. It also covers the components of model construction including state variables, parameters, equations, and the definition of volumetric and specific rates for microbial growth, death, product formation, and substrate uptake. The classification aims to account for heterogeneity at both the population and intracellular levels in modeling biological systems.
The B cell receptor is a transmembrane protein on B cells that is composed of a membrane-bound immunoglobulin molecule and a signal transduction moiety. The B cell receptor consists of an Ig molecule anchored to the cell's surface and has two key functions: signal transduction upon antigen interaction and internalization of antigens for processing and presentation to T cells. The B cell co-receptor is a complex of CD19, CD21, and CD81 expressed on mature B cells.
Enzymes use several catalytic mechanisms to lower the free energy of transition states and greatly increase reaction rates, including acid-base catalysis, covalent catalysis, metal ion catalysis, and bringing substrates into close proximity and proper orientation. Acid-base catalysis involves proton transfer from catalytic amino acid side chains. Covalent catalysis transiently forms covalent bonds between enzyme and substrate. Metal ion catalysis uses transition metals to orient substrates, mediate redox reactions, or stabilize charges. Proximity and orientation align substrates for reaction, while catalysis by approximation brings two substrates together for reaction.
Fluorescence recovery after photo bleachinganasshokor
FRAP (Fluorescence Recovery After Photobleaching) is a technique used to study the dynamics of molecules in living cells. It involves photobleaching a fluorescent marker in a region of the cell using a focused laser beam. The diffusion and movement of non-bleached fluorescent molecules into the bleached area is then monitored over time, allowing calculation of diffusion coefficients. Considerations in FRAP analysis include the degree of bleaching, bleach/recovery times, beam radius, and photobleaching artifacts. FRAP has various applications such as determining connectivity between cellular compartments using FLIP (Fluorescence Loss In Photobleaching) and quantifying transport between compartments.
Enzymes are macromolecular biological catalysts that are proteins. They catalyze reactions by lowering the activation energy. Enzymes have several key properties - they are catalytic, colloidal in nature, highly specific, sensitive to temperature and pH changes. Enzymes are classified based on the type of reaction they catalyze into six main classes. The factors that affect enzyme activity include enzyme and substrate concentration, temperature, pH, and product concentration. Enzyme activity is highest at its optimal temperature and pH.
Kinetics of multi substrate enzyme catalyzed reactionHina Qaiser
Enzyme kinetics is the study of enzyme-catalyzed chemical reactions. Enzymes lower the activation energy of reactions by binding substrates to their active sites. Multi-substrate reactions can follow sequential or non-sequential mechanisms. In sequential mechanisms, both substrates must bind before any product is released, while non-sequential mechanisms allow product release before all substrates bind. Ping-pong mechanisms are a type of non-sequential mechanism where the enzyme is temporarily modified between substrate bindings.
This document discusses gas chromatography, including its principles, instrumentation, and applications. Gas chromatography is a process that separates components in a mixture using an inert gas as the mobile phase. There are two main types - gas-solid chromatography uses a gas mobile phase and solid stationary phase, while gas-liquid chromatography uses a gas mobile phase and liquid stationary phase coated on an inert solid support. Key components of a gas chromatography instrument include the carrier gas, sample injection port, columns, and detectors such as the flame ionization detector. Gas chromatography has applications in quality control and analysis of drugs and metabolites.
Enzymes catalyze chemical reactions by reducing the activation energy needed for the reaction to occur. They do this using several mechanisms including acid-base catalysis, covalent bond formation, and metal ion catalysis. Enzymes are also able to increase reaction rates by properly orienting substrates. Enzyme activity can be inhibited through various reversible and irreversible mechanisms such as competitive inhibition where an inhibitor binds to the active site, and suicide inhibition where the inhibitor is converted by the enzyme into a tightly-binding form. The Michaelis-Menten model and Lineweaver-Burk plots are commonly used to study enzyme kinetics and inhibition types.
This document discusses various ways that enzyme activity can be regulated. It describes how enzyme levels and activity can be increased or decreased through different mechanisms, including substrate availability, product accumulation, allosteric effectors, covalent modification, genetic controls, zymogens, isozymes, and modulator proteins. A key example discussed is allosteric regulation, where binding of an allosteric effector at a site other than the active site can increase or decrease an enzyme's catalytic efficiency. Phosphorylation is provided as a common example of covalent regulation, where the addition of phosphate groups can alter an enzyme's activity.
activation energy of biological systemKAUSHAL SAHU
SOME GENERAL TERM
FREE ENERGY
ENDERGONIC REACTION
EXERGONIC REACTION
ACTIVATION ENERGY
DEFINITION
TRANSITION STATE
WHERE DOES ACTIVATION ENERGY COME FROM?
DETERMINING THE ACTIVATION ENERGY THROUGH ARREHINIUS EQUATION
EFFECTS OF TEMPERATURE ON ACTIVATION ENERGY
NEGATIVE ACTIVATION ENERGY
EFFECTS OF ENZYMES ON ACTIVATION ENERGY
CONCLUSION
REFERENCES
Ionophores are molecules that transport ions across biological membranes. They contain both hydrophilic regions that bind ions and hydrophobic regions that interact with membrane lipids. Ionophores are classified based on their mechanism of action as either mobile carrier ionophores which transport ion complexes, or channel-forming ionophores which introduce pores for ion passage. Examples include valinomycin which transports potassium ions, gramicidin A which forms channels for cation transport, and ionomycin which carries calcium ions into cells. Ionophores have important applications as antibiotics, in research to manipulate cellular physiology, and as feed additives to improve livestock growth and productivity.
This presentation is about the kinetics of enzyme action , the Michaelis- Menten Model and kinetics of allosteric enzyme action in a simplified language.
The document discusses hydrophobic interactions and effects. It defines hydrophobicity as molecules that do not interact well with water, such as nonpolar substances. When these molecules are introduced into water, they aggregate together to minimize contact with water molecules. This occurs because breaking hydrogen bonds between water and the hydrophobic molecules is entropically unfavorable. The hydrophobic effect is driven by this entropy change and causes nonpolar substances to cluster together in aqueous solutions.
The document summarizes fatty acid oxidation pathways. Fatty acids undergo beta-oxidation where they are sequentially shortened by two carbons into acetyl-CoA. This occurs through activation, transport into mitochondria via carnitine shuttle, and four steps of beta-oxidation. Alpha-oxidation removes the alpha carbon for certain fatty acids like phytanic acid to allow beta-oxidation. Abnormalities in fatty acid oxidation can cause clinical conditions.
This document provides an overview of enzymology and enzymes. It discusses how enzymes are biological catalysts that accelerate chemical reactions in living organisms. Each reaction is catalyzed by one or more specific enzymes, which are proteins that recognize substrate molecules and facilitate their transformation. Enzymes play a key role in coupling exergonic and endergonic reactions to allow biochemical processes to occur under the constraints of thermodynamics. The document covers basics of enzyme kinetics, cofactors, classification, factors influencing enzyme activity such as temperature and pH, inhibition, and measurement of enzymatic activity.
Cell adhesion molecules help cells stick to each other and their surroundings through proteins. There are several types of cell adhesion molecules including immunoglobulin super family CAMs, integrins, selectins, and cadherins. Cadherins like E-cadherin form adherens junctions between cells and link to actin through catenins. Changes in cell adhesion can lead to diseases such as cancer where reduced adhesion allows cancer cells to invade other tissues. Cell adhesion molecules are important for tissue development and function.
What is enzyme?
How enzyme catalyze the reaction
Enzyme kinetics
History
Enzyme kinetic equation
Michaelis-menten equation
Michaelis-menten curve
Michaelis-menten equation derivation
Reversible inhibition
Two substrate reaction
Conclusion
References
This document discusses cell growth and methods for quantifying cell concentration and mass. It explains that growth occurs through replication and changes in cell size in response to nutrients, which are used for energy production, biosynthesis, and increasing mass. Methods for quantifying cell concentration include direct counts using a hemocytometer or plate counts, as well as indirect particle counters. Cell mass can be determined directly through dry weight, packed cell volume, or optical density measurements, or indirectly by measuring substrate consumption or product formation.
This document describes the airlift bioreactor, which uses forced air circulation to mix cells and nutrients without mechanical agitation. It has an inner riser region where air is injected upwards, and an outer downcomer region where degassed media and cells circulate downwards. The density gradient between these regions drives continuous fluid circulation. The bioreactor has a gas separator, sparger, and headspace to introduce air, separate gases, and allow foaming. It is useful for culturing shear-sensitive cells as it provides gentle mixing with low energy use.
The immobilization of whole cells can be defined as “the physical confinement or localization of intact cells to a certain region of space, without loss of desired biological activity.”
In other words, cell immobilization means to freeze an entire cell in a state of suspended animation, such that its metabolism stops and hence does not die.
Biological films are the multilayer growth of cells on solid support surfaces ; community of micro-organisms enclosed in a polymeric matrix and adhered on inert or living surface
These attached cells are embedded in a self-produced exopolysaccharide matrix, and exhibit different growth and bioactivity compared with suspended cells.
Biofilm consists of three components:
microorganism, extracellular polymeric substances (EPS),
surface for attachment.
The excreted polymeric substances hold the biofilm together and cement it to a surface.
The thickness of a biofilm is an important factor affecting the performance of the biotic phase.
Thin biofilms - low rates of conversion due to low biomass concentration.
Thick biofilms - may experience diffusionally limited growth, which may or may not be beneficial depending on the cellular system and objectives
FPLC is a type of liquid chromatography where the flow rate of solvents is controlled by pumps to ensure constant flow. The sample is introduced into a column containing small gel beads as the stationary phase. As the solvent flows through the column driven by the pumps, the different components of the sample separate as they interact differently with the gel beads, forming separated bands of the sample components based on properties like molecular weight and charge. FPLC allows for fast, controlled purification of biomolecules using chromatography.
The document discusses enzyme kinetics and the Michaelis-Menten model. It explains that enzymes convert substrates to products over time in three phases: an initial rapid increase, then a steady state. The Michaelis-Menten model describes enzyme velocity as a function of substrate concentration using the constants KM and Vmax. KM represents the substrate concentration at half Vmax. Inhibitors are also discussed, including competitive and noncompetitive inhibition and how they affect the kinetic parameters.
The document describes enzyme kinetics and the Michaelis-Menten model. It explains that enzyme velocity follows a curve when graphed against substrate concentration. This curve can be linearized using a double reciprocal plot to determine the Michaelis constant (KM) and maximum velocity (Vmax). KM represents the substrate concentration at half maximal velocity and provides information about enzyme affinity. The document also discusses different types of enzyme inhibitors and how they alter the kinetic parameters.
Enzymes use several catalytic mechanisms to lower the free energy of transition states and greatly increase reaction rates, including acid-base catalysis, covalent catalysis, metal ion catalysis, and bringing substrates into close proximity and proper orientation. Acid-base catalysis involves proton transfer from catalytic amino acid side chains. Covalent catalysis transiently forms covalent bonds between enzyme and substrate. Metal ion catalysis uses transition metals to orient substrates, mediate redox reactions, or stabilize charges. Proximity and orientation align substrates for reaction, while catalysis by approximation brings two substrates together for reaction.
Fluorescence recovery after photo bleachinganasshokor
FRAP (Fluorescence Recovery After Photobleaching) is a technique used to study the dynamics of molecules in living cells. It involves photobleaching a fluorescent marker in a region of the cell using a focused laser beam. The diffusion and movement of non-bleached fluorescent molecules into the bleached area is then monitored over time, allowing calculation of diffusion coefficients. Considerations in FRAP analysis include the degree of bleaching, bleach/recovery times, beam radius, and photobleaching artifacts. FRAP has various applications such as determining connectivity between cellular compartments using FLIP (Fluorescence Loss In Photobleaching) and quantifying transport between compartments.
Enzymes are macromolecular biological catalysts that are proteins. They catalyze reactions by lowering the activation energy. Enzymes have several key properties - they are catalytic, colloidal in nature, highly specific, sensitive to temperature and pH changes. Enzymes are classified based on the type of reaction they catalyze into six main classes. The factors that affect enzyme activity include enzyme and substrate concentration, temperature, pH, and product concentration. Enzyme activity is highest at its optimal temperature and pH.
Kinetics of multi substrate enzyme catalyzed reactionHina Qaiser
Enzyme kinetics is the study of enzyme-catalyzed chemical reactions. Enzymes lower the activation energy of reactions by binding substrates to their active sites. Multi-substrate reactions can follow sequential or non-sequential mechanisms. In sequential mechanisms, both substrates must bind before any product is released, while non-sequential mechanisms allow product release before all substrates bind. Ping-pong mechanisms are a type of non-sequential mechanism where the enzyme is temporarily modified between substrate bindings.
This document discusses gas chromatography, including its principles, instrumentation, and applications. Gas chromatography is a process that separates components in a mixture using an inert gas as the mobile phase. There are two main types - gas-solid chromatography uses a gas mobile phase and solid stationary phase, while gas-liquid chromatography uses a gas mobile phase and liquid stationary phase coated on an inert solid support. Key components of a gas chromatography instrument include the carrier gas, sample injection port, columns, and detectors such as the flame ionization detector. Gas chromatography has applications in quality control and analysis of drugs and metabolites.
Enzymes catalyze chemical reactions by reducing the activation energy needed for the reaction to occur. They do this using several mechanisms including acid-base catalysis, covalent bond formation, and metal ion catalysis. Enzymes are also able to increase reaction rates by properly orienting substrates. Enzyme activity can be inhibited through various reversible and irreversible mechanisms such as competitive inhibition where an inhibitor binds to the active site, and suicide inhibition where the inhibitor is converted by the enzyme into a tightly-binding form. The Michaelis-Menten model and Lineweaver-Burk plots are commonly used to study enzyme kinetics and inhibition types.
This document discusses various ways that enzyme activity can be regulated. It describes how enzyme levels and activity can be increased or decreased through different mechanisms, including substrate availability, product accumulation, allosteric effectors, covalent modification, genetic controls, zymogens, isozymes, and modulator proteins. A key example discussed is allosteric regulation, where binding of an allosteric effector at a site other than the active site can increase or decrease an enzyme's catalytic efficiency. Phosphorylation is provided as a common example of covalent regulation, where the addition of phosphate groups can alter an enzyme's activity.
activation energy of biological systemKAUSHAL SAHU
SOME GENERAL TERM
FREE ENERGY
ENDERGONIC REACTION
EXERGONIC REACTION
ACTIVATION ENERGY
DEFINITION
TRANSITION STATE
WHERE DOES ACTIVATION ENERGY COME FROM?
DETERMINING THE ACTIVATION ENERGY THROUGH ARREHINIUS EQUATION
EFFECTS OF TEMPERATURE ON ACTIVATION ENERGY
NEGATIVE ACTIVATION ENERGY
EFFECTS OF ENZYMES ON ACTIVATION ENERGY
CONCLUSION
REFERENCES
Ionophores are molecules that transport ions across biological membranes. They contain both hydrophilic regions that bind ions and hydrophobic regions that interact with membrane lipids. Ionophores are classified based on their mechanism of action as either mobile carrier ionophores which transport ion complexes, or channel-forming ionophores which introduce pores for ion passage. Examples include valinomycin which transports potassium ions, gramicidin A which forms channels for cation transport, and ionomycin which carries calcium ions into cells. Ionophores have important applications as antibiotics, in research to manipulate cellular physiology, and as feed additives to improve livestock growth and productivity.
This presentation is about the kinetics of enzyme action , the Michaelis- Menten Model and kinetics of allosteric enzyme action in a simplified language.
The document discusses hydrophobic interactions and effects. It defines hydrophobicity as molecules that do not interact well with water, such as nonpolar substances. When these molecules are introduced into water, they aggregate together to minimize contact with water molecules. This occurs because breaking hydrogen bonds between water and the hydrophobic molecules is entropically unfavorable. The hydrophobic effect is driven by this entropy change and causes nonpolar substances to cluster together in aqueous solutions.
The document summarizes fatty acid oxidation pathways. Fatty acids undergo beta-oxidation where they are sequentially shortened by two carbons into acetyl-CoA. This occurs through activation, transport into mitochondria via carnitine shuttle, and four steps of beta-oxidation. Alpha-oxidation removes the alpha carbon for certain fatty acids like phytanic acid to allow beta-oxidation. Abnormalities in fatty acid oxidation can cause clinical conditions.
This document provides an overview of enzymology and enzymes. It discusses how enzymes are biological catalysts that accelerate chemical reactions in living organisms. Each reaction is catalyzed by one or more specific enzymes, which are proteins that recognize substrate molecules and facilitate their transformation. Enzymes play a key role in coupling exergonic and endergonic reactions to allow biochemical processes to occur under the constraints of thermodynamics. The document covers basics of enzyme kinetics, cofactors, classification, factors influencing enzyme activity such as temperature and pH, inhibition, and measurement of enzymatic activity.
Cell adhesion molecules help cells stick to each other and their surroundings through proteins. There are several types of cell adhesion molecules including immunoglobulin super family CAMs, integrins, selectins, and cadherins. Cadherins like E-cadherin form adherens junctions between cells and link to actin through catenins. Changes in cell adhesion can lead to diseases such as cancer where reduced adhesion allows cancer cells to invade other tissues. Cell adhesion molecules are important for tissue development and function.
What is enzyme?
How enzyme catalyze the reaction
Enzyme kinetics
History
Enzyme kinetic equation
Michaelis-menten equation
Michaelis-menten curve
Michaelis-menten equation derivation
Reversible inhibition
Two substrate reaction
Conclusion
References
This document discusses cell growth and methods for quantifying cell concentration and mass. It explains that growth occurs through replication and changes in cell size in response to nutrients, which are used for energy production, biosynthesis, and increasing mass. Methods for quantifying cell concentration include direct counts using a hemocytometer or plate counts, as well as indirect particle counters. Cell mass can be determined directly through dry weight, packed cell volume, or optical density measurements, or indirectly by measuring substrate consumption or product formation.
This document describes the airlift bioreactor, which uses forced air circulation to mix cells and nutrients without mechanical agitation. It has an inner riser region where air is injected upwards, and an outer downcomer region where degassed media and cells circulate downwards. The density gradient between these regions drives continuous fluid circulation. The bioreactor has a gas separator, sparger, and headspace to introduce air, separate gases, and allow foaming. It is useful for culturing shear-sensitive cells as it provides gentle mixing with low energy use.
The immobilization of whole cells can be defined as “the physical confinement or localization of intact cells to a certain region of space, without loss of desired biological activity.”
In other words, cell immobilization means to freeze an entire cell in a state of suspended animation, such that its metabolism stops and hence does not die.
Biological films are the multilayer growth of cells on solid support surfaces ; community of micro-organisms enclosed in a polymeric matrix and adhered on inert or living surface
These attached cells are embedded in a self-produced exopolysaccharide matrix, and exhibit different growth and bioactivity compared with suspended cells.
Biofilm consists of three components:
microorganism, extracellular polymeric substances (EPS),
surface for attachment.
The excreted polymeric substances hold the biofilm together and cement it to a surface.
The thickness of a biofilm is an important factor affecting the performance of the biotic phase.
Thin biofilms - low rates of conversion due to low biomass concentration.
Thick biofilms - may experience diffusionally limited growth, which may or may not be beneficial depending on the cellular system and objectives
FPLC is a type of liquid chromatography where the flow rate of solvents is controlled by pumps to ensure constant flow. The sample is introduced into a column containing small gel beads as the stationary phase. As the solvent flows through the column driven by the pumps, the different components of the sample separate as they interact differently with the gel beads, forming separated bands of the sample components based on properties like molecular weight and charge. FPLC allows for fast, controlled purification of biomolecules using chromatography.
The document discusses enzyme kinetics and the Michaelis-Menten model. It explains that enzymes convert substrates to products over time in three phases: an initial rapid increase, then a steady state. The Michaelis-Menten model describes enzyme velocity as a function of substrate concentration using the constants KM and Vmax. KM represents the substrate concentration at half Vmax. Inhibitors are also discussed, including competitive and noncompetitive inhibition and how they affect the kinetic parameters.
The document describes enzyme kinetics and the Michaelis-Menten model. It explains that enzyme velocity follows a curve when graphed against substrate concentration. This curve can be linearized using a double reciprocal plot to determine the Michaelis constant (KM) and maximum velocity (Vmax). KM represents the substrate concentration at half maximal velocity and provides information about enzyme affinity. The document also discusses different types of enzyme inhibitors and how they alter the kinetic parameters.
1) Chemical kinetics describes relationships between reaction rates and concentrations of reactants and products. Enzyme kinetics follows Michaelis-Menten kinetics which describes reaction rates at varying substrate concentrations.
2) The Michaelis-Menten equation models reaction rates as substrate concentration increases, with an initial linear increase until reaching the maximum reaction rate (Vmax) at substrate saturation.
3) The Michaelis constant KM represents the substrate concentration at half Vmax and indicates an enzyme's affinity for its substrate. Lower KM means higher affinity.
Enzyme kinetics, factors and mechanism of enzyme activityShubhrat Maheshwari
Enzyme kinetics involves studying the chemical reactions catalyzed by enzymes. There are two common kinetic processes: the Michaelis-Menten plot and Lineweaver-Burk plot. The Michaelis-Menten plot models the reaction between an enzyme, substrate, and product using an equation. It relates reaction rate to substrate concentration and determines values like Vmax and Km. The Lineweaver-Burk plot is a double reciprocal plot that modifies the Michaelis-Menten equation to allow determining Vmax and Km from the slope and y-intercept. Enzyme activity is affected by factors like concentration, temperature, pH, and activators. Mechanisms of enzyme action include the lock-and
This document summarizes key concepts about enzyme kinetics and regulation:
1) Enzyme kinetics follows Michaelis-Menten models where the initial reaction rate (V0) increases with substrate concentration until reaching the maximum rate (Vmax) when enzyme sites are saturated.
2) Values like Km, Vmax, and Kcat/Km characterize enzyme-substrate binding affinity and catalytic efficiency. Km is the substrate concentration when V0 is half Vmax.
3) Reactions can involve single or multiple substrates through sequential or ping-pong mechanisms. Allosteric enzymes have cooperative binding between subunits and may be regulated by pathway end-products.
This document discusses enzymes and enzyme kinetics. It defines enzymes as biomolecules that catalyze chemical reactions and provides examples of enzyme units and the SI unit for enzyme activity, the katal. The document then summarizes Michaelis-Menten enzyme kinetics, including the key assumptions of the MM model. It describes parameters of the MM equation like Km, Vmax, kcat, and kcat/Km. Finally, it discusses enzyme inhibition, categorizing inhibitors as reversible or irreversible and describing different types of reversible and irreversible inhibition.
The document discusses the Michaelis-Menten equation, which was devised in 1913 to explain the relationship between reaction velocity and substrate concentration in enzyme-catalyzed reactions. It is based on the assumption that the enzyme and substrate form a reversible enzyme-substrate complex in the initial step of the reaction. The Michaelis constant Km represents the substrate concentration at which the reaction velocity is half of its maximum value Vmax and can be used to measure the enzyme's affinity for the substrate. The Lineweaver-Burk plot is also described as a way to determine Km and Vmax values graphically from experimental data.
1) The document discusses enzyme kinetics and the Michaelis-Menten model of enzyme kinetics.
2) It introduces key concepts such as the Michaelis constant Km and maximum velocity Vmax, and derives the Michaelis-Menten equation relating substrate concentration, reaction rate, Km and Vmax.
3) It also discusses factors that affect enzyme activity such as temperature, pH, and enzyme inhibitors, distinguishing between competitive, uncompetitive, and noncompetitive inhibition.
This presentation deals with basics of enzyme kinetics and introduction to various plots which aid in understanding the mechanism of inhibition of enzymes.
Michaelis - Menten Curve of Enzyme Kinetic.pptxAkhil Pradeep
The document discusses enzyme kinetics and inhibition. It describes how the reaction rate increases with substrate concentration until the enzyme becomes saturated. It also discusses the Michaelis-Menten equation and how the Michaelis constant (Km) represents the substrate concentration required for half maximal velocity. Further, it describes competitive, uncompetitive, and noncompetitive inhibition and how they differ in their effects on Km and Vmax.
Enzymes are proteins that act as biological catalysts, increasing the rate of chemical reactions by lowering their activation energy. There are two main models that describe how enzymes bind substrates - the lock-and-key model and induced fit model. The Michaelis-Menten model describes enzyme kinetics, defining terms like the Michaelis constant KM and maximum reaction rate Vmax. Enzymes can be inhibited by inhibitors in competitive, noncompetitive, or uncompetitive manners, each affecting the enzyme kinetics differently as seen in Lineweaver-Burk plots.
Enzyme kinetics can provide information about enzyme activity under different conditions. The Michaelis-Menten approach models enzyme-catalyzed reactions and describes reaction rates with the Michaelis-Menten constant (Km) and maximum reaction rate (Vmax). Enzymes can be inhibited reversibly or irreversibly by inhibitors that reduce reaction rates. Different types of reversible inhibition include competitive, uncompetitive, and mixed inhibition. Temperature, pH, and allosteric effectors can regulate enzymatic activity through various mechanisms.
1) The document discusses key concepts related to enzyme kinetics including Michaelis-Menten kinetics, reaction orders, and kinetic parameters.
2) It derives the Michaelis-Menten equation based on the assumptions that the enzyme-substrate complex is in steady state and the rate limiting step is breakdown of this complex.
3) Important kinetic parameters discussed include Km, Vmax, Kcat, and Kcat/Km, which provide information about enzyme-substrate affinity and catalytic efficiency.
4) Limitations of the Michaelis-Menten model for enzymes exhibiting cooperative binding or more complex reaction mechanisms are also addressed.
Here you can find out the information about a very important topic of Biochemistry i.e. Enzyme Kinetics.
I have elaborated how enzymes kinetics works . The most important equation Michaelis-menten equation, and Burk plot.
#enzymes #biochemistry
The document discusses factors that affect the rate of enzyme action, including enzyme concentration, substrate concentration, temperature, pH, concentration of coenzymes and activators, time, and inhibitors. It provides details on how each factor influences the reaction rate. Specifically, it explains that enzyme activity is highest when substrate concentration is saturating but not excessive, and when other conditions like temperature and pH are optimal. The document also describes Michaelis-Menten kinetics and how reversible and irreversible inhibition can decrease reaction rates.
This document discusses factors that affect enzyme action and rates of enzyme reactions. It covers several key points:
1) Enzyme reaction rates depend on substrate and product concentrations, temperature, pH, the presence of activators or inhibitors, and time.
2) Most enzymes follow Michaelis-Menten kinetics, where the affinity of the enzyme for the substrate is represented by the Michaelis constant Km.
3) Temperature, pH, substrate and product concentrations can all influence the shape of curves describing enzyme kinetics like Michaelis-Menten. Optimal temperatures and pH levels vary between enzymes.
4) Methods like double reciprocal plots, Dixon plots, and the Hill equation are used to further study
Kinetics of Enzyme Action Enzyme kineticsAkhil Pradeep
1. Enzyme kinetics is the study of enzymatic reaction rates in response to experimental parameters like substrate concentration and inhibitors. It expresses chemical reactions mathematically.
2. In 1913, Michaelis and Menten postulated the existence of an enzyme-substrate complex and proposed a kinetic model where the enzyme binds substrate to form a complex, converts it to product, and releases product.
3. Their model derived a relationship between substrate concentration and reaction rate, known as the Michaelis-Menten equation, which produces a hyperbolic curve when graphed. This describes how reaction rate increases with substrate concentration until the enzyme becomes saturated.
This document provides an overview of enzyme kinetics. It defines important terms like rate constant, substrate concentration, enzyme unit, and Michaelis constant. It describes models used to study enzyme kinetics like the Michaelis-Menten equations and Lineweaver-Burk, Hanes-Woolf, and Eadie-Hofstee plots. It discusses factors that affect reaction rates like pH, temperature, and inhibitors. The document also covers cooperative enzyme systems and multireactant reactions.
ENZYME INHIBITION & FACTORS AFFECTING THE VELOCITY OF ENZYME ACTIONYESANNA
This document discusses several key factors that affect enzyme activity:
1. Enzyme and substrate concentration - Reaction rate increases with increasing concentrations of enzyme and substrate up to a maximum.
2. Temperature and pH - Enzymes have optimal temperatures and pH levels for activity. Outside these ranges, activity decreases.
3. Inhibitors and activators - Substances that bind enzymes can inhibit or activate their activity, altering reaction rates. Common types of inhibitors are competitive, non-competitive, and uncompetitive.
The document summarizes an experiment on the effects of substrate concentration, temperature, and pH on an enzyme reaction. It finds that a lower Km value indicates a higher affinity of the enzyme for the substrate. It also determines that increasing temperature increases the rate of reaction up to a point, and that most enzymes function best within a specific pH range, with activity lost outside this range due to breaking of bonds in the enzyme structure. The experiment is able to prepare standard reference graphs and investigate how the three factors influence the enzyme concentration as measured by the maximum reaction rate Vmax and Michaelis constant Km.
A bacteriophage is a virus that infects bacteria. Lambda phage is a temperate bacteriophage that has two life cycle choices: lytic and lysogenic. During lysogeny, the lambda repressor binds to the operator region (OR) on the phage DNA and represses transcription of lytic genes, allowing the phage genome to remain dormant as a prophage integrated into the bacterial chromosome.
The trp operon controls the biosynthesis of tryptophan in E. coli. It contains 5 genes that encode enzymes for tryptophan production. The operon uses attenuation to regulate expression based on tryptophan levels. When tryptophan is low, transcription proceeds through the leader sequence. When tryptophan is high, translation is rapid and a stem loop structure forms, terminating transcription. The trp operon is a repressible system, where the effector molecule allows the repressor to bind the operator and shut down expression.
The document summarizes the lac operon in E. coli, which controls the breakdown of lactose. The lac operon contains 3 genes - lacZ, lacY, and lacA - that code for enzymes involved in lactose catabolism. In the absence of lactose, a repressor protein binds to the operator region and prevents transcription. In the presence of lactose, it binds to the repressor and induces transcription of the structural genes. The lac operon demonstrates both negative control by the repressor and positive control through induction by lactose binding. Glucose also regulates the operon through catabolite repression involving cAMP levels.
Post-translational modifications (PTMs) are chemical changes that occur to proteins after translation. PTMs regulate protein activity, localization, and interactions. The main types of PTMs are phosphorylation, glycosylation, ubiquitination, and methylation. Phosphorylation involves the addition of phosphate groups and is important for cell signaling. Glycosylation adds carbohydrate groups and affects protein structure. Ubiquitination tags proteins for destruction, and methylation adds methyl groups, regulating processes like gene expression. PTMs are identified through techniques like mass spectrometry and chromatographic analysis.
Aminoglycosides like streptomycin bind to the 30S ribosomal subunit and interfere with initiation complex formation, inducing misreading of mRNA and breaking polysomes into monosomes. Chloramphenicol inhibits protein synthesis by binding reversibly to the 50S ribosomal subunit and preventing the binding of aminoacyl tRNA to the acceptor site. Tetracyclines also bind to the 30S ribosomal subunit but prevent the binding of aminoacyl tRNA to the mRNA ribosome complex. Macrolides inhibit protein synthesis by reversibly binding to the 50S ribosomal subunit and suppressing translocation of mRNA.
Protein synthesis involves three main steps - initiation, elongation, and termination. In initiation, the small and large ribosomal subunits assemble along with mRNA and tRNA to form the initiation complex. In elongation, amino acids are added one by one to the growing polypeptide chain. Termination occurs when a stop codon is reached, causing the release of the completed protein. While the overall process is similar between prokaryotes and eukaryotes, there are some key differences like the number of initiation factors and whether mRNA is polycistronic or monocistronic.
Ribosomes are organelles found in all cells that synthesize proteins. They consist of RNA and proteins and exist as two subunits - a smaller 30S subunit in prokaryotes and 40S in eukaryotes, and a larger 50S subunit in prokaryotes and 60S in eukaryotes. Ribosomes translate mRNA into proteins through initiation, elongation, and termination steps. Errors in ribosome functioning can lead to improper protein folding and diseases.
This document discusses genetic code, tRNA, and translation. It provides definitions of key terms like codon, anticodon, wobble hypothesis. It describes the structure and function of tRNA, including how it is charged with specific amino acids by aminoacyl tRNA synthetases. The document also discusses characteristics of the genetic code, including that it is degenerate, uses triplet codons, and has start and stop signals. It provides information on ribosomes, including their composition in prokaryotes and eukaryotes. In summary, the document provides an overview of the mechanisms and key components involved in translating genetic code into proteins.
Mutation is a change in genetic material that can be caused by errors during DNA replication or DNA repair. There are several types of mutations including point mutations, insertions, deletions, and chromosomal mutations. Point mutations include transitions, transversions, missense mutations, and nonsense mutations. Insertions and deletions can disrupt the genetic code. Spontaneous mutations arise naturally while induced mutations are caused by mutagens like radiation, chemicals, or viruses. Mutations can be germline or somatic and can have different effects on protein function and the phenotype. The document provides examples of specific mutations and their effects.
Telomeres are repetitive DNA sequences at the ends of chromosomes that protect them from degradation. Telomeres naturally shorten each time a cell divides until they reach a critical shortness that causes cell senescence. Telomerase is an enzyme that adds telomere repeats to chromosome ends and counteracts shortening. It is active in 90% of cancer cells, allowing unlimited cell division by maintaining telomere length, but is not generally active in most adult somatic cells.
This document discusses DNA replication and the central dogma. It covers the basic requirements for DNA replication including substrates, templates, enzymes, and primers. The stages of replication - initiation, elongation, and termination - are described. Key aspects of the replication process are explained, such as semi-conservative mechanism, unwinding of DNA, formation of replication forks, and bidirectional replication. Differences between prokaryotic and eukaryotic DNA replication are highlighted. Finally, various inhibitors of DNA replication are listed.
RNA differs from DNA in several key ways. RNA is typically single-stranded, contains ribose sugar instead of deoxyribose, and contains uracil instead of thymine. There are multiple types of RNA that serve different cellular functions, including messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). mRNA carries coding information from DNA to the ribosome for protein synthesis. tRNA transfers amino acids to the ribosome during protein assembly according to the mRNA codon sequence. rRNA is a core component of ribosomes and facilitates protein translation.
Transcriptional regulatory elements such as promoters, enhancers, silencers, and insulators help control gene expression. Promoters initiate transcription and contain core and proximal elements. Enhancers can activate transcription from farther distances by binding activator proteins. Silencers negatively regulate genes by binding repressor proteins. Insulators block interactions between genes to prevent neighboring transcriptional effects. These cis-acting elements help precisely regulate protein levels through transcriptional mechanisms.
Ribozymes are RNA molecules that act as enzymes and catalyze biochemical reactions. They were first discovered in 1982 by Thomas Czech and Sidney Altman, who later won the Nobel Prize in Chemistry for their discovery. Ribozymes increase the rate and specificity of reactions like phosphodiester bond cleavage and peptide bond synthesis. Common types of ribozymes include self-splicing introns, RNase P, hammerhead ribozymes, and hairpin ribozymes. Artificial ribozymes can also be synthesized in the laboratory by mutating natural ribozymes.
Rifampicin binds to the beta subunit of prokaryotic RNA polymerase, inhibiting prokaryotic transcription initiation. It selectively binds bacterial RNA polymerase without affecting eukaryotic polymerases. This allows rifampicin to be an effective treatment for bacterial infections like tuberculosis and leprosy. Alpha amanitin from death cap mushrooms potently inhibits RNA polymerase II during both transcription initiation and elongation, potentially causing death in 10 days from just one mushroom due to failure of gene expression.
Eukaryotic pre-mRNA undergoes processing in the nucleus before being exported to the cytoplasm for protein synthesis. This involves adding a 5' cap and poly-A tail to increase stability and facilitate export. Introns are also spliced out by the spliceosome, a complex of small nuclear RNAs and proteins that cuts out introns and joins exons to form mature mRNA. Capping occurs at the 5' end shortly after transcription, while polyadenylation adds around 200 adenine nucleotides to the 3' end. Splicing removes intervening intron sequences by cutting and religating exons. These processing steps produce translation-competent mRNA from initial pre-mRNA transcripts.
Transcription is the first step in gene expression for eukaryotic organisms where DNA is copied into RNA. This process involves RNA polymerase binding to promoter regions on DNA and synthesizing a complementary RNA strand. Transcription results in RNA transcripts that can then undergo further processing and modification before being translated into proteins.
The document summarizes transcription in prokaryotes. It discusses the key components including the template strand, coding strand, and RNA polymerase. RNA polymerase is made up of multiple subunits and recognizes promoter sequences to initiate transcription. The process of transcription involves three phases - initiation when RNA polymerase binds to the promoter, elongation as the RNA strand continuously grows, and termination when RNA polymerase stops synthesis.
DNA is a double-helix molecule that carries genetic instructions. It is composed of two strands called polynucleotides made up of nucleotides, each containing a nucleobase (A, T, C, or G), sugar, and phosphate. The strands are stabilized by hydrogen bonds between nucleotides and base stacking. DNA can be denatured by heat, pH extremes, or chemicals, breaking the hydrogen bonds and separating the strands. Denaturation temperature depends on factors like composition, length, and environment. Renaturation occurs when strands reconnect under appropriate conditions.
DNA's double helical structure is stabilized by several weak forces that collectively provide strong stabilization. Hydrogen bonding between complementary base pairs provides some stability, while base stacking interactions between the hydrophobic bases, including hydrophobic and van der Waals forces, provide additional stability by burying the bases in the interior. Ionic interactions between the negatively charged phosphate backbone and positive ions like magnesium also contribute to stability. Though each individual interaction is weak, the collective effects of all of these forces interacting along the entire DNA molecule strongly stabilize its double helical structure.
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Enzyme technology solved problems
1. ENZYME TECHNOLOGY –
SOLVED PROBLEMS
Dr.Anant Achary and Dr.S.Karthikumar
Kamaraj College of Engineering and Technology
S.P.G.C.Nagar, K.Vellakulam, Madurai Dist. TN, INDIA
skarthikumar@gmail.com
3. • Reactions can be independent of the concentration of substrate (0-
order), directly dependent on substrate (1st-order) or dependent on
substrate concentration raised to some higher power (2, etc.).
A.1
5. Km
• Km = Michaelis constant. It is the substrate concentration that gives
1/2 Vmax. That is, it is the concentration of substrate at which half
the active sites are filled. Km is also related to rate constants of the
individual steps in the reaction.
A.2
9. • kcat = the turnover number and is equal to k2 in the Michaelis
Menten mechanism.
• The turnover number of an enzyme is equal to the number of moles
of substrate converted to product per minute per mole of enzyme
present when the enzyme is fully complexed with substrate.
A.4
10. • Km is all too frequently equated with Ks. In fact, in most reactions
there is an appreciable disparity between the values for Km and Ks.
For the reaction A B define conditions under which Km = Ks.
Describe conditions under which this is not true
Q.5
12. • What is the steady-state approximation, and under what conditions is
it valid?
Q.6
13. The steady state approximation assumes that the concentrations of the
intermediates in a reaction do not change while the rate of product
formation is being measured. This holds for the early stages of a
reaction, after the ES complex has formed and before appreciable
changes have occurred in either the substrate or product
concentrations.
A.6
14. True or False
a. At saturating levels of substrate, the rate of an enzyme catalysed
reaction is proportional to the enzyme concentration.
b. The Michaelis constant Km equals the substrate concentration at which
v = 1/2 Vmax.
c. The Km for a regulatory enzyme varies with enzyme concentration.
d. If enough substrate is added, the normal Vmax of an enzyme catalysed
reaction can be attained even in the presence of a noncompetitive
inhibitor.
Q.7
15. e. The Km of some enzymes may be altered by the presence of
metabolites structurally unrelated to the substrate.
f. The rate of an enzyme-catalyzed reaction in the presence of a rate-
limiting concentration of substrate decreases with time.
g. The sigmoidal shape of the v versus (S) curve for some regulatory
enzymes indicates that the affinity of the enzyme for substrate
decreases as the substrate concentration is increased.
Q.7 Cont.
16. • a. True. Vmax = k2[E]t
• b. True
• c. False. The value of Km is independent of enzyme concentration for almost all enzymes
• d. False. A non-competitive inhibitor cannot be overcome by substrate concentration.
• e. True. This occurs in regulatory enzymes.
• f. True.
• g. False. The initial increasing slope of the curve shows that binding of the first substrate
molecule increases the affinity of the enzyme for subsequent substrate molecules.
A.7
17. 1. The _____________ of a reaction is the numerical relationship between
substrates and products
2. The rate constant ____________ of an enzyme-catalysed reaction is a measure
of the catalytic efficiency at saturating
levels of substrate.
3. _______________ inhibitors do not alter the Vmax of an enzyme-catalyzed
reaction.
4. The sigmoidal shape of the v versus [S] curve for some regulatory enzymes
results from a _______________ effect of substrate on the substrate binding sites.
5. For an enzyme whose Km can be regulated, the presence of a _____________
effector increases the level of substrate required to attain a given reaction rate.
Q.8
19. Assume that an enzyme catalyzed reaction follows Michaelis Menten
kinetics with a Km of 1 x 10-6 M. If the initial reaction rate is 0.1
μmol/min at 0.1 M, what would it be at 0.01 M, 10-3 M, and 10-6 M?
Q.9
21. A more general form of an equation for an enzyme catalyzed
reaction is:
Consider the essentially irreversible reaction represented by the free energy
diagram below.
A. Using the letters indicated in the diagram, relate each of the rate
constants in the Equation above to the energy-level difference that
determines it.
B. Which rate constant limits the rate of formation of product?
C. Does Km approximately equal Ks for this enzyme?
Q.10
22. A. The rate constant for each step is inversely related to the difference between the energy level of
the reactants and the highest energy barrier between the reactants and the products of that step. In
terms of the letters in the Figure,
k1 is determined by b-a
k-1 is determined by b-c
k2 is determined by d-c
k-2 is determined by d-e
k3 is determined by f-e, and
k-3 is determined by f-g.
B. k2 corresponds to a much higher energy barrier than the other forward rate constants and
therefore must limit the rate of product formation.
c. Since k2 is small relative to k-1 , Km approximates Ks for this enzyme. Therefore, Km is a measure
of affinity for substrate
A.10
23. To study the dependence of the rate of an enzyme-
catalyzed reaction on the substrate concentration, a
constant amount of enzyme is added to a series of
reaction mixtures containing different
concentrations of substrate (usually expressed in
mol/L).
The initial reaction rates are determined by
measuring the number of moles (or μmoles) of
substrate consumed (or product produced) per
minute. Consider such an experiment in which the
initial rates in Table were obtained at the indicated
substrate concentrations.
Initial rates at various substrate concentrations for a
hypothetical enzyme-catalyzed reaction
a. What is Vmax for this reaction?
b. Why is v constant above substrate concentrations of 2.0 x
10-3 M?
c. What is the concentration of free enzyme at 2.0 x 10-2 M
substrate concentration?
Q.11
24. a. Vmax = 60μmol/min
b. v is constant because it has reached Vmax; the enzyme is saturated
with substrate.
c. The concentration of free enzyme is negligible because all of the
enzyme is in the ES form.
A.11
25. To study the dependence of the rate of an enzyme-catalyzed
reaction on the substrate concentration, a constant amount of
enzyme is added to a series of reaction mixtures containing
different concentrations of substrate (usually expressed in mol/L).
Total reaction mixture volume is 10mL
The initial reaction rates are determined by measuring the
number of moles (or μmoles) of substrate consumed (or product
produced) per minute. Consider such an experiment in which the
initial rates in Table were obtained at the indicated substrate
concentrations.
A. What is Vmax for this concentration of enzyme?
B What is the Km of this enzyme?
C. Show that this reaction does or does not follow simple
Michaelis-Menten kinetics.
D. What are the initial rates at [S] = 1.0 x 10-6 M and at
[S] = 1.0 x 10-1 M?
E. Calculate the total amount of product made during the first
five minutes when [S] = 2.0 x 10-3 M. Could you make the
same calculation at [S] = 2.0 X 10-6 M?
F. Suppose that the enzyme concentration in each reaction
mixture were increased by a factor of 4. What would be the
value of Km? of Vmax? What would be the value of v at [S]
= 5.0 x 10-6 M?
Q.12
26. A. Vmax = 0.25 μmol/min
B. For a reaction obeying Michaelis-Menten kinetics, Vmax and Km are
simply constants relating v to [S]. Km can be calculated by substituting
Vmax and any pair of v and [S] values at v < Vmax. For example, at [S] =
5.0 x 10-5 M and v = 0.20 μmol/min the equation becomes
A.12
27. C. If the reaction follows simple Michaelis-Menten kinetics, then the
Michaelis-Menton equation should relate v to [S] over a wide range of [S].
This can be tested by determining whether the equation yields the same
value of Km at several different values of [S] and v < Vmax. Under the
conditions of this problem, the same value, Km = 1.3 x 10-5 M, is obtained at
[S] = 5.0 x 10-6 M, v = 0.071 μmol/min and at [S] = 5.0 x 10-7 M, v= 0.0096
μmol/min. Therefore, Michaelis-Menten kinetics are obeyed
A.12. Cont.
29. E. At [S] = 2.0 x 10-3 M, v = Vmax = 0.25 μmol/min. Since 0.25 μmole is much less than the amount
of substrate present (2.0 x 10-3 mole/liter x 10-2 L x 106 μmol/mol = 20 μmol) the reaction can
proceed for five minutes without significantly changing the substrate concentration.
Thus, 0.25 μmol/min x 5 min = 1.25 μmol
At [S] = 2.0 x 10-6 M,
During 5 minutes at this rate, 0.033 μmol/min x 5 min = 0.17 μmol of product would be produced.
However, this value exceeds the total amount of substrate present (2.0 x 10-6 mol/L x 10-2 L x 106
μmol/mol = 0.020 μmol). Clearly, during the-5 min reaction, [S] and therefore v would decrease
significantly. Calculation of the exact amount of product made would require integration of a
differential equation; this amount obviously cannot exceed 0.020 μmole
A.12 Cont.
30. F. Km is independent of enzyme concentration, since a change in [E]
does not affect the three rate constants, k1, k2, and k3.
Hence Km would remain equal to 1.25 x 10-5 M.
Since Vmax = k3[E]o, increasing the enzyme concentration by a factor
of 4 increases Vmax by a factor of 4. Therefore, Vmax = 1.0 μmole/min.
At [S] = 5.0 x 10-6 M,
A.12 Cont.
31. The Km of a certain enzyme is 1.0 X 10-5 M in a reaction that is described by Michaelis-Menten kinetics. At a
substrate concentration of 0.10 M, the initial rate of the reaction is 37 μmol/min for a certain concentration of
enzyme. However, you observe that at a lower substrate concentration of 0.010 M the initial reaction rate remains 37
μmoles/min.
• a. Using numerical calculations, show why this tenfold reduction in substrate concentration does not alter the
initial reaction rate.
• b. Calculate v as a fraction of Vmax for [S] = 0.20 Km, 0.50 Km, 1.0 Km, 2.0 Km, 4.0 Km, and 10 Km.
• c. From the results in (b), sketch the curve relating v/Vmax to [S]/Km. What is the best range of [S] to use in
determining Km or investigating the dependence of v on [S]?
Q.13
32. a. Since both substrate concentrations are well above Km, you
can assume that Vmax = 37 μmol/min. Then
Therefore, at [S] 1.0 x 10-2 M, v still is equal to Vmax.
A.13
33. b. From the Michaelis-Menten
equation, the following
relationships can be calculated:
A.13 Cont.
34. c. When you plot these values you will be able to see that the best
range of [S] for studying the dependence of v on [S] is in the
neighborhood of Km or below it, since changes in [S] below Km cause
greater changes in v than do changes in [S] above Km. Therefore, when
using graphic methods to determine Km and Vmax, several
measurements should be made at [S] well below Km.
A.13 Cont.
35. The hydrolysis of pyrophosphate to orthophosphate is important in driving forward biosynthetic
reactions such as the synthesis of DNA. This hydrolytic reaction is catalyzed in E. coli by a
pyrophosphatase that has a mass of 120 kDa and consists of six identical subunits.
Purified enzyme has a Vmax of 2800 units per milligram of enzyme. For this enzyme, a unit of
activity is
defined as the amount of enzyme that hydrolyzes 10 μmol of pyrophosphate in 15 minutes at 37°C
under standard assay conditions.
A. How many moles of substrate are hydrolyzed per second per milligram of enzyme when the
substrate concentration is much greater than Km?
B. How many moles of active site are there in 1 mg of enzyme? Assume that each subunit has one
active site.
C. What is the turnover number of the enzyme?
Q.14
36. a.1 'unit' here = 10 μmole/15 min at 37ºC
= 10/15 μmole min-1
= 10/15.60 μmole s-1
When [S] >> Km,
Vo = Vmax
Here Vmax = 2800 units mg-1
= 2800 x 10/15.60
= 31.3 μmol s-1 mg-1.
NOTE. THIS IS BY DEFINITION THE SP. ACT.
A.14
37. b. 1mg of enzyme = 10-3/MW moles = 103/MW μmoles
= 103/120000
= 1/120 μmoles.
But each mole of enzyme has 6 active sites. Therefore 1 mg of enzyme
=1/120 x 6
= 1/20
= 0.05 μmoles active site.
A.14 Conti.
38. c. The units of sp. act. are, μmoles (unit time)-1 (mg enzyme)-1. Here μmoles s-1
mg-1 is used, and the value is 31.3 μmole s- 1 mg-1.
The turnover number, kcat, is effectively the activity in terms of (μmole active site)-
1, instead of (mg enzyme)-1 as in sp. act., and its units are,
μmoles s-1 (μmole active site)-1 = s-1.
We know from '(b)' that there are 0.05 μmoles active site per
mg of enzyme, i.e.,
μmole of active site = 1/0.05
= 20 mg of enzyme, and that the sp. act. (activity per mg of enzyme)
= 31.3 μmoles s-1 mg-1.
Therefore the kcat (activity per mmole of active site, i.e. its activity per 20 mg of
enzyme) is = 31.3 x 20
= 626 s-1.
A.14 Conti.
39. In the cases of severe liver damage, an enzyme EL
is released into the blood. After severe exercise,
an isozyme from muscle, EM, is found in the
blood. EL and EM can be differentiated since they
have different kinetic constants. The Km of the
liver enzyme is 3 x 10-4 M; the Km of the muscle
enzyme is 7 x 10-5 M.
Data from assays on an unconscious patient's
blood are given below. Ten microliters of blood
was used in each assay.
a. Is the patient likely to be suffering from a liver disease or had
she been exercising too strenuously?
b. Explain your reasoning for your answer to part a.
Q.15
40. • a. Liver
• b. The [S]Vmax/2 is close to the Km of the liver enzyme
A.15
41. Define the following
A. a unit of enzyme activity
B. steady-state conditions
c. oligomeric enzyme
Q.16
42. A. The amount of enzyme which converts 1 μmole of substrate to
product per min (at 25/37oC)
B. Under steady state conditions, S is converted into P at a constant
rate, the [S] and [P] vs time plots are linear, and [ES] is Constant
C. An enzyme with a quaternary structure, i.e., made up of more than
one subunit (protomer, monomer). The subunits may be the same
(e.g., a homodimer), or different (e.g., a heterodimer).
A.16
43. Under what experimental conditions does an enzyme-catalysed
reaction follow zero-order kinetics?
Q.17
45. • Indicate the effects of substrate concentration, enzyme
concentration, temperature, inhibitors or activators on enzyme
activity by labeling correctly both axes of the graphs given shown
below
Your choice for axes are: Energy, [E], Temperature, [S], 1/[S], 1/v, v.
(You may use the same label on more than one graph.)
Q.18
47. An enzyme-catalyzed reaction was assayed at several substrate
concentrations. Two data points which fell on the Lineweaver- Burk plot
are v = 41.7 μmol S/min when [S] = 5 x 10-4 M and v = 16.7 μmol S/min
when [S] = 5 x 10-6 M. Place the two points on a line on the
accompanying graph.
A. Determine the value of Km and Vmax in the correct units. When an
inhibitor was added, the velocities fell to 1/2 their uninhibited values.
B. Plot the inhibited line on the graph.
C. Is the inhibitor competitive or non-competitive? On what evidence
did you base your decision?
Q.19
48. • a. Km = 8.3 x 10-6 M, Vmax = 45.5 μmoles min-1
• c. Non-competitive (Km same, Vmax smaller)
A.19
49. Fumarase (L-malate hydrolase) catalyzes the reversible hydration of fumarate to L-malate. The fumarase from pig heart has been
crystallized (M.W. 197,000 Da) and consists of four subunits that can be dissociated into inactive monomers under relatively mild
conditions. Substrate can induce reformation of tetramers with complete recovery of the activity. The subunits have a molecular
weight of 48,500 Da and each contains three free –SH groups. Fumarase requires no cofactors. Kinetic studies implicate the
participation of a pair of groups on the enzyme (one acidic, one basic) with pKa's of 6.2 and 6.8. These groups have been postulated
to be two imidazole groups of histidine residues, one in the imidazole form and one in the imidazolium form. The reverse reaction is
stereospecific for L-malate and only Lmalate is produced from fumarate.
A. For fumarase, an intact ___________ structure is necessary.
B. Predict graphically the effect of pH on the activity of fumarase. At what pH
would you expect maximal enzyme activity?
C. Malonate, -OOC-CH2-COO- is an inhibitor of fumarase. What type of
inhibitor would you expect malonate to be?
D. Using a Lineweaver-Burk plot, graphically illustrate the effect of malonate
on the kinetics of fumarase. Be sure to label all parts of the graph as well as
the inhibitor data-
Q.20
50. A. Quaternary (oligomeric)
B. pH 6.5
C. Competes with fumarate because of similar structure, therefore
a competitive inhibitor
A.20
51. A. What is meant by an allosteric enzyme?
B. What is the difference between the active site and the regulatory
site of an allosteric enzyme?
C. How does an allosteric inhibitor produce its effect on an enzyme?
Q.21
52. A. They are enzymes whose kinetic properties cannot be accounted for
by the Michaelis-Menton model.
B. The active site is where the substrate binds to the regulatory
enzyme; the regulatory site is where the effector molecules bind. These
two sites are different.
C. An allosteric inhibitor typically binds and stabilizes the enzyme in an
inactive or less active (conformation) state.
A.21
53. A. The activation energy for a non enzyme-catalyzed reaction is
_____________ than the activation energy for the same reaction
catalyzed by an enzyme.
B. Why would increasing the temperature of an enzyme-catalysed
reaction from 25°C to 37°C increase the rate of the reaction?
C. Why would increasing the temperature to 60°C probably cause a
decrease in the rate of the enzyme-catalyzed reaction?
Q.22
55. A. If the concentration of substrate is 10-3 M and the concentration of
enzyme is 10-8 M, what would be the effect of the observed rate of
production of product if the enzyme concentration were doubled?
(Assume Km is 10-6 M.)
B. What would be the effect on the observed rate if the substrate
concentration were doubled to 2 x 10-3 M but the enzyme
concentration remained at 10-8 M?
Q.23
56. A. [S] >> Km
Therefore, vo = Vmax Therefore, the velocity is doubled when [E]tot is
doubled
B. None, because the enzyme is already saturated with substrate
A.23
57. Answer the following with true or false; justify your answer in each
case.
A. The initial rate of an enzyme-catalyzed reaction is independent of
substrate concentration.
B. If enough substrate is added, the normal Vmax of an enzyme
catalyzed reaction can be attained even in the presence of a
noncompetitive inhibitor.
C. The rate of an enzyme-catalyzed reaction in the presence of a rate-
limiting concentration of substrate decreases with time.
D. The sigmoid shape of the v-versus-[S] curve for some regulatory
enzymes indicates that the affinity of the enzyme for substrate
decreases as [S] is increased
Q.24
59. Two forms of isocitric dehydrogenase exist in mammals. One which is NAD+ specific and found
only in mitochondria and a NADP+ specific enzyme found in both cytosol and mitochondria
NADP+ specific isocitric dehydrogenase catalyzes the decarboxylation by formation of an
unstable enzyme bound chelate of Mn2+ and a a-keto acid intermediate. The free energy change
for formation of aketo glutaric acid under physiological conditions is: DG° = -5 kcal/mol. AMP
regulates the enzymatic activity by reducing Km for isocitrate by 10 fold. Only {isocitrate2-} was
found to be the substrate form that binds to the enzyme.
Q.25
60. A. Mn2+ is an example of a(an)
B. NAD+ is an example of a(an); The reaction velocity was found to be 4th order with respect to isocitric acid indicating
high cooperativity.
C. The fact that "high cooperativity "is found indicates that this enzyme is a(an) ______________ enzyme. The NAD+
specific enzyme has a molecular weight of 330,000 Daltons and is made up of 8 identical subunits.
D. The NAD+ specific enzyme is an example of a(an) ______________ protein and the level of structural organization for
the 8 identical subunits is termed the structure of the protein.
E. AMP is said to act as a(an) Reaction velocity is decreased in presence of ATP which acts by binding at the same site
that NAD+ binds.
F. ATP is said to act as a(an) ____________
G. Production of NADH during the course of the reaction would be expected to ____________ the reaction velocity and
NADH would be an example of a(an) ____________.
H. What is the significance of DGo = -5 kcal/mole in terms of:
1. The desire of the reaction to go as written? (One sentence answer).
2. The activation energy of the reaction?
I. If isocitrate ionization was the only important controlling factor for the pH-activity profile of the reaction. (See
characteristics at the beginning of this exam). Using the information given, construct an accurate pH-activity profile for
the enzyme catalyzed reaction. Use graph paper.
Q.25 cont.
61. A. Cofactor
B. Coenzyme
C. Allosteric (positive Km-type)
D. Oligomeric
E. Quaternary
F. +ve heterotropic effector
G. -ve heterotropic effector
H. Equal, coenzyme
I.
1. If NAD+/NADP+ and NADH /NADPH and isocitrate and a-ketoglutarate all at 1 M concentration are
mixed at 1 atm pressure, since the free energy change under standard conditions for the forward (L R)
reaction is negative, NAD+/NADP+ and isocitrate will be converted into NADH/NADPH and a-glutaric acid.
(Also, at equilibrium, the ratio of products to reactants (Keq), will be greater than
1, i.e., the equilibrium lies to the right.)
2. The standard free energy change in the L R direction is the difference between the standard free
energies of activation in the forward and reverse directions (DG0’ = DG*for - DG*rev)
A.25
62. Zero order kinetics in an enzyme catalyzed reaction only occurs when
we have:
a. a high specific activity
b. an isozyme present
c. high substrate concentration
d. a high Km
e. high enzyme concentration
f. a high turnover number
Q.26
64. The redox pairs NAD+ NADH and NADP+ NADPH are well suited for use
in coupled clinical enzyme assay systems:
a. because of their acid-base properties.
b. because of their distinctly different absorbance properties of their
oxidized and reduced species.
c. because of their occurrence in cells.
d. because of their ability to take the place of enzyme reactions.
e. because they are coenzymes.
Q.27
66. The success of a measurement of an enzyme activity using a coupled
enzyme assay depends on having
a. The NAD+ NADH dehydrogenase reaction.
b. the second reaction as non-limiting.
c. A non-buffered reaction medium.
d. a colorimeter adequate in the visible region of the spectrum.
e. a trained M.D. to supervise.
Q.28
68. In measuring the rate of a coupled reaction one must know:
a. how to control the humidity in the sample chamber.
b. where the lag phase ends.
c. the maximum absorbance of the unknown enzyme.
d. the rate of absorbance change during the preincubation period.
e. the total absorbance change throughout the assay.
Q.29
70. The levels of LDH isozymes in the blood are indicative of certain disease states: Some of the
isozymes present will react with a particular substrate while others will not. Thus, one can
experimentally measure the activity of these particular isozymes while other isozymes are also
present in the sample. In particular, this assay makes particular use of:
a. the colligative properties of the solution.
b. the substrate specificity of the isozyme of interest.
c. the preincubation phase of the isozyme of interest.
d. the tertiary structure of the isozymes of interest.
e. the total activity of all enzymes species.
Q.30
72. Let's say the following assay was established to measure E1 levels in
serum.
a. List the solution conditions you would have to control to make this a valid assay.
b. List the species to equation 1 and 2 whose concentrations you would have to manipulate in order to
make a valid assay.
c. One of your technicians ran the assay under conditions which, with normal serum levels of E1, the
Vmax for equation
(1) turned out to be about equal to Vmax for equation
(2). State what is wrong with the assay.
d. In one sentence state what you would do to rectify the problem.
Q.31
73. a. The standard conditions of assay for E1 must be met (pH, ionic strength, cofactors,
temperature, etc), so the results may be compared to the range of normal values.
b. [A] must be at a sufficiently high concentration to saturate E1. [NAD+] must be at a
sufficiently high concentration to saturate E2. E2 must be present at sufficiently high levels
of activity for it not to be a limiting factor in the assay
c. The rate of the second reaction must be much greater than that of the first reaction (E2
at high activity), so that only the amount of E1 limits the rate of the reaction, i.e., Vmax2
must be >> Vmax1.
d. Increase the activity of E2 present in the assay medium, so that it is much greater than
that expected for E1.
A.31
74. Clinical data for enzymes are often reported in terms of international
units. What is an enzyme unit of activity and what relationship does it
bear to specific (enzyme) activity?
Q.32
75. The IU is the amount of enzyme converting 1 μmole of S into P per min
(at a given temperature, pH etc). The specific activity = IU per mg total
protein (it increases as the enzyme is purified)
A.32
76. What is meant by the term "zero order" reaction as it pertains to
enzymes
Q.33
77. When the enzyme is saturated with substrate,
v0 = Vmax = kcat Et = a constant for that assay,
i.e., the rate = a constant = k, therefore it is not dependent on [S], and
zero order kinetics in [S} are observed.
A.33
78. Why is the preincubation phase of a coupled enzyme assay necessary?
Q.34
79. To take into account:
Breakdown of NADH/NADPH due to
a. Its intrinsic chemical instability
b. The effect of other factors in our serum sample on NADH levels
besides the enzyme we want to measure.
A.34
80. Which one of the following
relationships best describes how
reaction velocity can be used to
indicate the level of enzyme in
blood serum as developed for
clinical labs?
Q.35
81. d. Vmax = k [Eo] (i.e., Vmax = kcat [E]tot)
A.35