Name: graphing enzyme kinetics data
Uploaded: 2023-04-30T10:09:42.000Z
Duration: 1 min 19 s
Channel: JoVE

K12 Standards

north-carolina-k-12-science-standard-course-of-study

North Carolina K-12 Science Standard Course of Study

9-12

Biology

from-molecules-to-organisms-structures-and-processes

From Molecules to Organisms- Structures and Processes

LS.Bio.1.2

Introduction to Enzyme Kinetics

Ферментативная кинетика изучает скорость реакций, катализируемых ферментами. Частота повторных экспериментов при различных концентрациях субстрата контролируется путем измерения концентрации потребляемого субстрата или продукта, образующегося с течением времени.
Эти результаты можно изобразить на графике, чтобы показать, как концентрация субстрата влияет на скорость или скорость реакции.
Скорость реакции линейно увеличивается с увеличением количества субстрата при низких концентрациях, но при более высоких концентрациях она начинает стабилизироваться. Скорость приближается к максимальной скорости или Vmax – скорости, при которой фермент полностью насыщается субстратом.
Сродство фермента измеряет, насколько сильно или слабо фермент связывается со своим субстратом, и количественно измеряется KM, постоянной Михаэлиса. Значение KM равно концентрации субстрата при скорости 50% от Vmax.
Маленькая буква KM указывает на то, что фермент обладает высоким сродством к субстрату, и наоборот. Фермент с большим KM требует более высоких концентраций субстрата для приближения к своей максимальной скорости по сравнению с ферментом с более низким KM.

<ol>
	<li>Воет Д. и Воет Д.Г. Биохимия, 4-е изд. Джон Уайли и сыновья, 2010 стр. 487-492</li>
	<li>Берг Д.М., Тимочко Й.Л., и Страйер Л. Биохимия, 5-е изд., W.H. Freeman and Co., раздел 8.4</li>
</ol>

Введение в кинетику ферментов

Кинетика ферментов изучает скорость биохимических реакций. Ученые контролируют скорость реакции конкретной ферментативной реакции при различных концентрациях субстрата. Также могут быть проведены дополнительные испытания с ингибиторами или другими молекулами, влияющими на скорость реакции. 

 Затем экспериментатор может построить график начальной скорости или скорости реакции (Vo) данного испытания в зависимости от концентрации субстрата ([S]), чтобы получить график свойств реакции. Для многих ферментативных реакций с участием одного субстрата эти данные соответствуют уравнению Михаэлиса-Ментен, выведенному Леонором Михаэлис и Мод Ментен. 

 <img alt="Eq1" src="/files/ftp_upload/12164/12164_Equation 1.png" style="text-align: center; height: 70px;"> 

 Уравнение определяет максимальную скорость (Vmax) и константу Михаэлиса (KM) для изучаемого фермента и основано на следующих предположениях: 

 <ol> 
 	<li>В начале реакции продукт отсутствует.</li> 
 	<li>Скорость образования фермент-субстратного комплекса равна скорости диссоциации и распада на продукты.</li> 
 	<li>Концентрация фермента минимальна по сравнению с концентрацией субстрата.</li> 
 	<li>Измеряются только начальные скорости реакций.</li> 
 	<li>Фермент присутствует либо в свободной форме, либо в составе фермент-субстратного комплекса.</li> 
 </ol> 
 Различные модификации уравнения Михаэлиса-Ментен, такие как графики Лайнуивера-Берка, Иди-Хофсти и Хейнса-Вульфа, являются альтернативными способами построения графиков кинетических параметров. График Лайнуивера-Берка или график обратных двойных величин часто используется для оценки KM и Vmax. На графике используются обратные значения осей X и Y из графика Михаэлиса-Ментен. Математически точка пересечения по оси Y равна 1/Vmax, а точка пересечения по оси X равна -1/KM. 

 График Лайнуивера-Берка можно использовать для визуального различия типов ингибиторов: конкурентных, неконкурентных и неконкурентных. Различные перестановки уравнения Михаэлиса-Ментен, такие как графики Иди-Хофсти и Хейнса-Вульфа, также используются для определения кинетических параметров.

Кинетика ферментов изучает скорость биохимических реакций. Ученые следят за скоростью реакции для той или иной ферментативной реакции на различных субстратах ...

Education

JoVE Core

Cell Biology

Клеточная биология

Unit 1

Раздел 1

Energy and Catalysis

Энергетика и катализ

KEY TERMS AND CONCEPTS

ОСНОВНЫЕ ТЕРМИНЫ И ПОНЯТИЯ

graphing enzyme kinetics data

Enzyme kinetics uses graphs to show how fast a biochemical reaction happens. Scientists measure the initial reaction rate, or velocity (Vo), at different substrate concentrations ([S]). They may also run trials with inhibitors or other molecules that change the reaction rate.

The results are often plotted as Vo against [S]. For many single-substrate reactions, the data fits the Michaelis-Menten equation. This equation, named after Leonor Michaelis and Maud Menten, helps estimate the maximum velocity (Vmax) and the Michaelis constant (KM) for the enzyme being studied.

The Michaelis-Menten model is based on a few assumptions. No product is present at the start of the reaction. The rate of enzyme-substrate complex formation equals the rate of its breakdown into products and its dissociation. The enzyme concentration is minimal compared with the substrate concentration. The model also uses only the initial reaction rates. In this setup, the enzyme is either free or bound in an enzyme-substrate complex.

Scientists can also use other graph forms to analyze the same data. The Lineweaver-Burke plot, also called the double reciprocal plot, uses the reciprocal values from the Michaelis-Menten graph. On this plot, the y-intercept equals 1/Vmax and the x-intercept equals -1/KM. It is often used to estimate KM and Vmax and to visually distinguish competitive, non-competitive, and uncompetitive inhibitors.

Other rearrangements of the Michaelis-Menten equation include the Eadie-Hofstee plot and the Hanes-Woolf plot. These graphs are also used to determine kinetic parameters. Together, these methods help scientists compare reaction behavior and understand how enzymes respond to substrate concentration and inhibitors.

Graphing Enzyme Kinetics Data

the first law of thermodynamics

Energy Changes in Cells and Matter

The first law of thermodynamics describes how energy changes form in the universe. The total amount of energy stays constant. Energy can move from one place to another or change into a different form, but it cannot be created or destroyed. These energy changes happen in everyday life. A light bulb changes electrical energy into light energy. A gas stove changes chemical energy in natural gas into heat energy. Plants also carry out a major energy transformation when they convert sunlight energy...

the second law of thermodynamics

Entropy and Spontaneous Change

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an introduction to free energy

Gibbs Free Energy and Reaction Work

Gibbs free energy describes the usable energy in a chemical reaction. It helps scientists compare how much energy different reactions release. This measurement is called Gibbs free energy, or G, after Josiah Willard Gibbs. The second law of thermodynamics explains why not all energy stays available for work. Some energy is lost in an unusable form, such as heat, and this loss increases entropy. Gibbs free energy is the energy left after that entropy loss is taken into account. Every chemical...

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the equilibrium binding constant and binding strength

the-equilibrium-binding-constant-and-binding-strength

How Equilibrium Binding Constant Affects Binding

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free energy and equilibrium

Free Energy and Reaction Direction

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enzymes and activation energy

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Enzymes lower the energy needed for a chemical reaction to begin. That starting energy is called activation energy, or free energy of activation, and it is often written as Ea. During a reaction, bonds break and new bonds form. In glucose breakdown, for example, bonds between carbon atoms break and release energy because they are energy-storing bonds. Before a reaction can move forward, reactant molecules must reach a high-energy, unstable shape called the transition state. To get there, the...

introduction to enzyme kinetics

Enzyme kinetics uses graphs to show how fast a biochemical reaction happens. Scientists measure the initial reaction rate, or velocity (Vo), at different substrate concentrations ([S]). They may also run trials with inhibitors or other molecules that change the reaction rate. The results are often plotted as Vo against [S]. For many single-substrate reactions, the data fits the Michaelis-Menten equation. This equation, named after Leonor Michaelis and Maud Menten, helps estimate the maximum...

turnover number and catalytic efficiency

How Fast Enzymes Work in Digestion

Enzymes can work at very different speeds, and their turnover number shows how fast they convert substrate molecules into products. Turnover number is the maximum number of substrate molecules an enzyme can transform per unit time. For most enzymes, this value is between 1 and 1000 molecules per second. Some enzymes are especially fast. Catalase has the highest known turnover number and can convert up to 2.8×10^6 molecules of hydrogen peroxide into water and oxygen each second. Lysozyme has...

catalytically perfect enzymes

Diffusion-Controlled Enzymes and Reaction Speed

Catalytically perfect enzymes are enzymes that speed up biochemical reactions at very high rates. The idea was first proposed by W. J. Albery and J. R. Knowles in 1976. These enzymes have catalytic efficiency values of about 10^8 to 10^9 M^-1 s^-1 and are also called diffusion-controlled enzymes because diffusion of the substrate into the active site is the only rate-limiting step. Examples of catalytically perfect enzymes include triose phosphate isomerase, fumarase, and superoxide dismutase.

introduction to mechanisms of enzyme catalysis

introduction-to-mechanisms-of-enzyme-catalysis

How Enzymes Change Shape to Catalyze

Enzyme catalysis depends on how enzymes bind substrates and how the active site responds during the reaction. Scientists once described this process with a simple lock-and-key model, where the enzyme and substrate fit together in one instant. Research now supports induced fit, a more dynamic model in which binding causes a small change in the enzyme’s shape. That shift helps create an ideal match between the enzyme and the substrate’s transition state, which improves catalysis. The active site...

ligand binding and linkage

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cooperative allosteric transitions

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ribozymes

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atp energy storage and release

ATP Powers Cellular Transport and Reactions

ATP powers cellular transport and many reactions by transferring energy through phosphorylation. ATP is a highly unstable molecule. If it is not used quickly, it breaks down into ADP and inorganic phosphate, and the released energy is lost as heat. Cells use ATP through energy coupling. This means the exergonic reaction of ATP hydrolysis is linked to an endergonic reaction so the reaction can move forward. One major example is the sodium-potassium pump, a transmembrane ion pump that is vital...

atp and macromolecule synthesis

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ATP helps cells build biological macromolecules. These organic compounds are mainly made of carbon atoms bonded to hydrogen, oxygen, nitrogen, and small amounts of other elements. The four major classes of biological macromolecules are carbohydrates, lipids, proteins, and nucleic acids. Most macromolecules start as small subunits called monomers. Cells join monomers with covalent bonds to make larger molecules called polymers. This building process needs energy, so cells depend on ATP, the...

coupled reactions

ATP Powers Cellular Work

ATP powers many kinds of cellular work by coupling energy-releasing reactions to energy-requiring ones. Some reactions happen on their own and release energy. Others need an energy input to move forward. Cells link these reactions so important processes can still occur. Adenosine triphosphate, or ATP, stores energy that cells can use quickly. ATP hydrolysis is the breaking of ATP, and it releases energy. Cells often use that released energy to drive endergonic reactions, which are reactions...

key terms and concepts

Learn how enzyme kinetics data is graphed with Michaelis-Menten, Lineweaver-Burke, Eadie-Hofstee, and Hanes-Woolf plots to estimate Vmax and KM.

Graphing Enzyme Kinetics Data | JoVE K12

cell biology

JoVE Core Cell Biology illustrates complex cellular processes in easy-to-understand yet comprehensive animated video lessons. This collection focuses on the essential components and functions of cells, including organelles and membranes, cellular transport, and cell energetics. It is complemented by scientist-in-action videos that demonstrate the application of cell biology in real-world research experiments.

Virginia  Standards of Learning

HIgh School

BIO.2

BIO.2.c

Chemistry

CH.7

CH.7.f

Florida Department of Education

High School

Life Science

SC.912.L.18

SC.912.L.18.4

SC.912.L.18.11

Texas Essential Knowledge and Skills

BIO.11

BIO.11.B

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Nucleic Acid Structure

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LS.Bio.1.1

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Enzyme kinetics studies the rates of biochemical reactions. Scientists monitor the reaction rates for a particular enzymatic reaction at various substrate ...

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Ligand Binding and Linkage

Cooperative Allosteric Transitions

Ribozymes

Carry out investigations to illustrate how enzymes act as catalysts for biochemical reactions and how environmental factors affect enzyme activity.

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Prokaryotic Cells

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Cytoplasm

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The Nucleus

The nucleus is a membrane-bound organelle that acts as a control center in a eukaryotic cell. It contains chromosomal DNA, which controls gene expression ...

Endoplasmic Reticulum

Endoplasmic ReticulumThe endoplasmic reticulum (ER) is an extensive network of membranous sacs and tubules in eukaryotic cells, continuous with the outer ...

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Analyze how the relationship between structure and function supports life processes within organisms.

Graphing Enzyme Kinetics Data

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