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

酵素動態学は、酵素触媒反応の速度を研究します。さまざまな基質濃度での反復実験の速度は、消費された基質または時間の経過とともに形成された製品の濃度を測定することによって監視されます。
これらの結果をグラフ化して、基質濃度が反応の速度や速度にどのように影響するかを示すことができます。
反応速度は、低濃度では基質量の増加に伴って直線的に増加しますが、高濃度では頭打ちになり始めます。この速度は、最大速度またはVmax(酵素が基質と完全に飽和する速度)に近づきます。
酵素親和性は、酵素がその基質にどれだけ強くまたは弱く結合するかを測定し、ミカエリス定数であるKMによって定量されます。KMの値は、レートがV maxの50%のときの基板濃度に等しくなります。
KMが小さい場合は、酵素の基質親和性が高いことを示し、その逆も同様です。KMが大きい酵素は、KMが低い酵素と比較して、最大速度に近づくためにより高い基質濃度を必要とします。

<ol>
	<li>Voet D. and Voet J.G. Biochemistry, 4th ed. John Wiley & Sons, 2010, pp-487-492</li> (英語)
	<li>Berg J.M.、Tymoczko J.L.、およびStryer L. Biochemistry、第5版、WH Freeman and Co.、セクション8.4</li>
</ol>

酵素反応速度論の概要

酵素反応速度論は生化学反応の速度を研究します。 科学者は、さまざまな基質濃度での特定の酵素反応の反応速度を監視します。 反応速度に影響を与える阻害剤やその他の分子を使用した追加の試験も実行される場合があります。

実験者は、基質濃度 ([S]) に対して特定の試行の初期反応速度または速度 (V_o) をプロットして、反応特性のグラフを得ることができます。 単一の基質が関与する多くの酵素反応では、このデータは、レオノール・ミカエリスとモード・メンテンによって導かれた式であるミカエリス・メンテン式に適合します。

<img alt="Eq1" src="/files/ftp_upload/12164/12164_Equation 1.png" style="text-align: center; height: 70px;" />

この方程式は、研究対象の酵素の最大速度 (V_max) とミカエリス定数 (KM) を推定し、次の仮定に基づいています。

<ol>
	<li>反応開始時には生成物は存在しない。</li>
	<li>酵素と基質の複合体の形成速度は、生成物への解離と分解の速度に等しい。</li>
	<li>酵素濃度は基質濃度と比較してごくわずかである。</li>
	<li>初期反応速度のみが測定される。</li>
	<li>酵素は遊離型または酵素-基質複合体のいずれかで存在する。</li>
</ol>

ラインウィーバー＝バークプロット、イーディー＝ホフステープロット、ヘインズ＝ウルフプロットなど、ミカエリス-メンテン式のさまざまな再配置は、動力学的パラメーターをグラフ化する別の方法です。 ラインウィーバー＝バークまたは二重逆数プロットは、KM と V_max を推定するためによく使用されます。 このプロットでは、ミカエリス-メンテンプロットの x 軸と y 軸の逆数値が使用されます。 数学的には、y 切片は 1/V_max に等しく、x 切片は -1/KM に等しくなります。

ラインウィーバー＝バークプロットを使用すると、阻害剤のタイプ (競合的、非競合的、および非競合的) を視覚的に区別することができます。 イーディー＝ホフステープロット、や ヘインズ＝ウルフプロットなど、ミカエリス-メンテン式のさまざまな再配置も、動力学的パラメータを決定するために使用されます。

酵素速度論は、生化学反応の速度を研究します。科学者は、さまざまな基質での特定の酵素反応の反応速度を監視します...

Education

JoVE Core

Cell Biology

細胞生物学

Unit 1

ユニット1

Energy and Catalysis

エネルギーと触媒

KEY TERMS AND CONCEPTS

主要用語と概念

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

Entropy helps predict whether a process will happen on its own. Entropy is a measure of randomness or disorder in a system. High entropy means high disorder and low energy. A student’s bedroom is a simple example. If no work is done, the room becomes messy over time. That messy state is highly disordered and has high entropy. To make the room clean again, energy must be added as the student puts things away. That organized state has low entropy. Processes that increase the entropy of the...

enthalpy within the cell

Cellular Enthalpy and Energy Changes

Cellular enthalpy describes how heat and energy change during chemical and physical processes. Enthalpy is written as H and is defined as the sum of a system’s internal energy, U, and the product of pressure and volume, PV. It is a state function, which means its value depends on the state of the system, not the path taken. Scientists cannot measure the enthalpy of a single substance directly. They can determine enthalpy changes, or ΔH, for a process instead. For reactions or changes that...

entropy within the cell

Cell Energy Loss and Entropy

Cell energy loss and entropy explain why living things need constant energy input. Cells obtain energy, transform it, and use it to do work, but these transfers are never completely efficient. In each transfer, some energy is lost in a form that cannot be used. In many cases, that lost energy is heat energy.

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...

endergonic and exergonic reactions in the cell

endergonic-and-exergonic-reactions-in-the-cell

Cell Reactions That Release or Store Energy

Cell reactions can either release energy or store it for later use. In chemistry, this difference is described by the value of ΔG, or free energy change. When energy is released during a reaction, ΔG is negative. The products then have less free energy than the reactants because some free energy has been given off. Reactions with a negative ΔG are called exergonic reactions. Exergonic means energy is leaving the system. These reactions are also called spontaneous reactions because they can...

the equilibrium binding constant and binding strength

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

How Equilibrium Binding Constant Affects Binding

The equilibrium binding constant describes how strongly two molecules bind at equilibrium. It is a useful way to compare binding strength in molecular interactions. This topic focuses on how binding is measured and what the equilibrium binding constant means. A higher value indicates stronger binding, while a lower value indicates weaker binding. Understanding this constant helps explain how tightly molecules stay together under equilibrium conditions. The page also connects the binding...

free energy and equilibrium

Free Energy and Reaction Direction

Free energy and reaction direction are linked in chemical equilibrium. A free energy change, ΔG, shows the driving force for a process. When ΔG is negative, the process is driven forward. When ΔG is positive, the reverse direction is favored. When ΔG is zero, the forward and reverse driving forces are equal, and the system is at equilibrium. The reaction quotient, Q, helps show how close a system is to equilibrium. Q is the numerical value of the mass action expression for the system. It can...

non-equilibrium in the cell

Cells Stay Out of Equilibrium

Cells stay out of chemical equilibrium by constantly using energy and moving materials in and out. Chemical reactions in metabolism are often reversible. They can go in both directions. In one direction, they release energy to the surroundings. In the reverse direction, they absorb energy. The same idea applies to reactions that build and break down cell molecules. Proteins, for example, can be broken into individual amino acids, and amino acids can be used to build proteins again. In a closed...

oxidation and reduction of organic molecules

oxidation-and-reduction-of-organic-molecules

Electron Carriers in Cell Energy Transfer

Cell energy production depends on redox reactions, which are oxidation and reduction reactions that happen together. In oxidation, an electron is removed from an atom in a compound. In reduction, that same electron is added to another compound. Because these reactions usually occur as a pair, they are called redox reactions. When a molecule loses an electron, its potential energy goes down. The electron does not stay free in the cytoplasm. Instead, it moves to a second compound and reduces...

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Enzyme Function, Cofactors, and Inhibitors

Enzymes are proteins that speed up biochemical reactions without being used up. Humans have used enzymes since about 7000 BCE, when they fermented sugars to make alcohol without knowing the reaction was enzyme-catalyzed. The word enzyme was coined by Wilhelm Kuhne in 1877. It comes from the Greek words en, meaning “in” or “within,” and zyme, meaning “yeast.” Enzymes work through active sites, which are regions that bind substrates and help convert them into products. Many enzymes also have...

enzymes and activation energy

How Enzymes Lower Reaction Energy

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

Ligand Binding and Allosteric Effects

Ligand binding and linkage describe how one molecule can affect another when it binds to a protein. In this topic, the focus is on how binding at one site can influence activity at a different site on the same protein. This idea is important for understanding protein function in high school biology. It helps explain why some proteins respond more strongly after a ligand attaches. The transcript centers on the connection between ligand binding and linkage, which is a key concept in molecular...

cooperative allosteric transitions

How Proteins Shift Shape Together

Proteins can change shape in a coordinated way during allosteric transitions. In a cooperative transition, one part of the protein affects another part, so the change spreads through the molecule. These shape changes help proteins switch between different states. The shift can influence how the protein works and how it responds to other molecules. Cooperative allosteric transitions are an important idea in protein function. They explain how a small change at one site can lead to a larger...

ribozymes

Catalytic RNA: Ribozymes in Action

Ribozymes are RNA molecules that can act as enzymes. They help speed up specific chemical reactions without being protein enzymes. This makes them an important example of RNA with more than one job in the cell. Some ribozymes can cut RNA molecules, and others can join them together. This shows that RNA can do catalytic work as well as carry genetic information. Ribozymes are a key part of how scientists think about the many roles of RNA in biology. One well-known ribozyme is ribonuclease P,...

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

ATP in Building Cell Macromolecules

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 細胞生物学は、複雑な細胞プロセスを、わかりやすく包括的なアニメーション ビデオ レッスンで説明します。このコレクションは、細胞小器官や膜、細胞輸送、細胞のエネルギー学など、細胞の必須コンポーネントと機能に焦点を当てています。これは、実際の研究実験における細胞生物学の応用を実証する科学者による実際のビデオによって補足されています。

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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Fibrous Proteins

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LS.Bio.1.1

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Enzymes and Activation Energy

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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 ...

Turnover Number and Catalytic Efficiency

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The theory of catalytically perfect enzymes was first proposed by W.J. Albery and J. R. Knowles in 1976. These enzymes catalyze biochemical reactions at ...

Introduction to Mechanisms of Enzyme Catalysis

For many years, scientists thought that enzyme-substrate binding took place in a simple &#34;lock-and-key&#34; fashion. This model stated that the enzyme ...

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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Cells are the smallest and basic units of life, whether it is a single cell that forms the entire organism, e.g., in a bacterium or trillions of them, ...

Cell Size

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Prokaryotic Cells

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Cytoplasm

The cytoplasm consists of organelles and a framework of protein scaffolds called the cytoskeleton suspended in an aqueous solution, the cytosol. The ...

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

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Ribosomes

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Golgi Apparatus

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Lysosomes

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LS.Bio.1.3

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

Graphing Enzyme Kinetics Data

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