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

K12 Standards

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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

La cinetica enzimatica studia i tassi di reazioni catalizzate da enzimi. I tassi di esperimenti ripetuti a concentrazioni variabili di substrato vengono monitorati misurando la concentrazione di substrato consumato o di prodotto formato nel tempo.
Questi risultati possono essere rappresentati graficamente per mostrare come la concentrazione del substrato influenzi la velocità o la velocità di una reazione.
La velocità di reazione aumenta linearmente con l'aumentare della quantità di substrato a basse concentrazioni, ma inizia a stabilizzarsi a concentrazioni più elevate. La velocità si avvicina a una velocità massima o Vmax, la velocità in cui l'enzima è completamente saturo con il substrato.
L'affinità enzimatica misura la forza o la debolezza con cui un enzima lega il suo substrato ed è quantificata da KM, la costante di Michaelis. Il valore di KM è uguale alla concentrazione del substrato quando la velocità è del 50% del Vmax.
Un KM piccolo indica che un enzima ha un'elevata affinità per il substrato e viceversa. Un enzima con un KM maggiore richiede concentrazioni di substrato più elevate per avvicinarsi alla sua velocità massima rispetto a un enzima con un KM inferiore.

<ol>
	<li>Voet D. e Voet J.G. Biochimica, 4a ed. John Wiley & Sons, 2010 pp-487-492</li>
	<li>Berg J.M., Tymoczko J.L. e Stryer L. Biochimica, 5a ed., W.H. Freeman and Co., Sezione 8.4</li>
</ol>

introduzione alla cinetica degli enzimi

La cinetica enzimatica studia la velocità delle reazioni biochimiche. Gli scienziati monitorano le velocità di reazione per una particolare reazione enzimatica a varie concentrazioni di substrato. Possono essere eseguite anche ulteriori prove con inibitori o altre molecole che influenzano la velocità di reazione.

Lo sperimentatore può quindi tracciare il tasso di reazione iniziale o velocità (Vo) di una determinata prova rispetto alla concentrazione del substrato ([S]) per ottenere un grafico delle proprietà della reazione. Per molte reazioni enzimatiche che coinvolgono un singolo substrato, questi dati si adattano all'equazione di Michaelis-Menten, un'equazione derivata da Leonor Michaelis e Maud Menten.

<img alt="Eq1" src="/files/ftp_upload/12164/12164_Equation 1.png" style="text-align: center; height: 70px;" />

L'equazione stima la velocità massima (Vmax) e la costante di Michaelis (KM) per l'enzima studiato e si basa sui seguenti presupposti:

<ol>
	<li>Nessun prodotto è presente all'inizio della reazione.</li>
	<li>La velocità di formazione del complesso enzima-substrato è uguale alla velocità di dissociazione e scomposizione nei prodotti.</li>
	<li>La concentrazione dell'enzima è minima rispetto alla concentrazione del substrato.</li>
	<li>Vengono misurate solo le velocità di reazione iniziali.</li>
	<li>L'enzima è presente sia nella forma libera che nel complesso enzima-substrato.</li>
</ol>

Diverse riorganizzazioni dell'equazione di Michaelis-Menten, come i grafici Lineweaver-Burke, Eadie-Hofsteot e Hanes-Woolf, sono modi alternativi per rappresentare graficamente i parametri cinetici. Il grafico Lineweaver-Burke o doppio reciproco viene spesso utilizzato per stimare il KM e il Vmax. Il grafico utilizza i valori reciproci degli assi xey del grafico di Michaelis-Menten. Matematicamente, l'intercetta y è uguale a 1/Vmax e l'intercetta x è uguale a −1/KM.

Il grafico Lineweaver-Burke può essere utilizzato per differenziare visivamente i tipi di inibitori: competitivi e non competitivi. Per determinare i parametri cinetici vengono utilizzati anche diversi riarrangiamenti dell'equazione di Michaelis-Menten, come i grafici di Eadie-Hofstee e Hanes-Woolf.

Introduzione alla cinetica degli enzimi

La cinetica enzimatica studia i tassi di reazioni biochimiche. Gli scienziati monitorano le velocità di reazione di una particolare reazione enzimatica in vari substrati ...

Education

Didattica

JoVE Core

Cell Biology

Biologia cellulare

Unit 1

Unità 1

Energy and Catalysis

Energia e Catalisi

KEY TERMS AND CONCEPTS

TERMINI E CONCETTI CHIAVE

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

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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...

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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

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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

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BIO.11

BIO.11.B

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Graphing Enzyme Kinetics Data

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