Enzyme Kinetics Overview

Podcast

Play an AI-generated podcast conversation about this lesson

Questions and Answers

What happens to the rate of an enzyme reaction as the temperature increases beyond its optimum level?

It remains constant.

It increases until denaturation occurs.

It increases exponentially.

It decreases due to enzyme denaturation. (correct)

How does the concentration of enzyme affect the reaction velocity?

Reaction velocity decreases.

It is inversely proportional.

It has no effect.

It is directly proportional. (correct)

What is the optimum pH range for most enzymes?

6-8 (correct)

1-2

4-5

10-11

Which of the following statements about activators is true?

Metal ions can act as activators for certain enzymes. Signup and view all the answers

What characterizes a metalloenzyme?

It holds the metal tightly. Signup and view all the answers

What does the rate law express in relation to chemical reactions?

The speed of a reaction based on reactants' concentrations and a rate constant Signup and view all the answers

What is the primary focus of enzyme kinetics?

The measurement of rates of enzyme-catalyzed reactions and influencing factors Signup and view all the answers

How do enzyme-catalyzed reactions behave as substrate concentration increases?

They eventually exhibit saturation behavior and reach a maximum rate Signup and view all the answers

What is the initial observation made by Adrina Brown regarding enzyme kinetics?

Substrate concentration affects the rate only when substrate concentration is low Signup and view all the answers

Which terms are commonly used to model catalytic efficiency in enzyme kinetics?

Km, Vmax, and kcat Signup and view all the answers

What type of reactions does the rate law apply to?

Any chemical reactions, including those not involving enzymes Signup and view all the answers

What characterizes a first-order reaction in the context of the rate law?

The rate is proportional to the concentration of one reactant only Signup and view all the answers

What does the term 'enzyme-substrate complex' refer to?

The binding of the enzyme with the substrate before conversion to product Signup and view all the answers

What is the predicted size of the PCR product from the TA cloning of the VrFBP gene?

1023 bp Signup and view all the answers

Which polymerase is used for the PCR in the TA cloning of VrFBP gene?

HF Pfu DNA Polymerase Signup and view all the answers

What process follows the ligation in the construction of the recombinant plasmid?

Blue-white selection Signup and view all the answers

What is the role of T4 ligase in recombinant plasmid construction?

To ligate DNA fragments together Signup and view all the answers

What is the purpose of Dpn I digestion in the plasmid construction process?

To remove methylated plasmid Signup and view all the answers

What type of chromatography is used for protein purification in the described process?

Ion exchange chromatography Signup and view all the answers

What is the composition of Vr-cFBPase according to the provided information?

341 aa, 8 helices, and 16 β-strands Signup and view all the answers

During the TA cloning process, which selection method is employed to identify successful clones?

Blue-white selection Signup and view all the answers

What is the significance of A-tailing in the PCR process?

To facilitate binding to the vector Signup and view all the answers

What type of cell is indicated for transformation in the process described?

E. coli (DH5α) Signup and view all the answers

What initial step is taken to create a cDNA library from mung bean mRNA?

Reverse transcription Signup and view all the answers

Which type of cloning is indicated for the VrFBP gene in the content provided?

TA cloning Signup and view all the answers

What induces protein expression in the PET32-FBP vector system?

IPTG Signup and view all the answers

Which component of the protein structure allows for specific interactions with substrates?

Active site Signup and view all the answers

What effect did the replacement of Met251 by Leu have on enzyme performance?

It improved substrate affinity and activity. Signup and view all the answers

Which amino acid is crucial for maintaining the AMP binding conformation?

Asp32 Signup and view all the answers

What is the primary challenge associated with the use of soluble enzymes in industrial applications?

High cost of isolation and purification. Signup and view all the answers

What defines an immobilized enzyme?

An enzyme that is attached to an inert, insoluble material. Signup and view all the answers

What is a benefit of enzyme immobilization?

Enhanced stability under various conditions. Signup and view all the answers

Which characteristic of enzymes limits their widespread use in industries?

Their instability and the cost of recovery. Signup and view all the answers

What process is described as confining enzyme molecules to a distinct phase from the substrates and products?

Enzyme immobilization. Signup and view all the answers

What environmental factors can immobilized enzymes provide increased resistance to?

pH, temperature, and several environmental changes. Signup and view all the answers

What is the purpose of the Nde I and BamH I restriction enzymes in the construction of the PET32-FBP plasmid?

To create sticky ends for gene insertion Signup and view all the answers

What is the consequence of performing site-directed mutagenesis on the PET32-FBP plasmid?

It alters the amino acid sequence of the expressed protein Signup and view all the answers

What is the primary role of T4 ligase in plasmid construction?

To ligate the gene fragment into the vector Signup and view all the answers

Which of the following represents the catalytic efficiency of the enzyme as defined by Kcat/Km?

1.09 $ ext{μM s}^{-1}$ Signup and view all the answers

What does the term 'turnover number' (Kcat) refer to?

The maximum amount of substrate converted per unit time Signup and view all the answers

What effect does the AMP inhibitory constant (Ki) represent regarding enzyme activity?

The concentration at which AMP reduces enzyme activity by 50% Signup and view all the answers

Which step is crucial for verifying the successful transformation of the PET32-FBP-DH5α plasmid?

Colony PCR Signup and view all the answers

What is the significance of using methylated plasmid in site-directed mutagenesis?

It prevents digestion by Dpn I enzyme after PCR Signup and view all the answers

What does the term 'temperature sensitive' imply regarding the enzyme used in the experiments?

The enzyme works best at a specific temperature range Signup and view all the answers

What is the predicted size of the product in the colony PCR?

1248 bp Signup and view all the answers

Which of the following would NOT be a step in the protein expression process outlined?

Mutation of the gene sequence Signup and view all the answers

What does the presence of a supercoil in plasmid DNA indicate?

The plasmid is preparing for replication Signup and view all the answers

What is the role of ion exchange chromatography in the purification of proteins?

To isolate proteins based on charge Signup and view all the answers

What type of protein is most likely being expressed in the PET32-FBP plasmid system?

Enzyme Signup and view all the answers

Study Notes

Enzyme Kinetics Summary:

Enzyme kinetics studies the rates of enzyme-catalyzed reactions, focusing on factors influencing these rates.
This field began in 1902 with Adrina Brown's work on invertase, which demonstrated that reaction rate can become independent of sucrose concentration when sucrose concentration is much higher than that of the enzyme.
Key elements of enzyme kinetics:
- Enzymes (E) bind to substrates (S) to form an enzyme-substrate complex (ES).
- The ES complex converts to a product (P) while regenerating the enzyme (E).
- The focus is on how enzymes accelerate reactions, how substrate concentration influences reaction rate, and modeling catalytic efficiency.
- Key terms include Km, Vmax, and kcat.
Factors affecting enzyme reaction rate:
- Substrate concentration:
  - Higher concentration leads to more collisions and a faster reaction rate.
- Enzyme concentration:
  - Higher enzyme concentration leads to a directly proportional increase in reaction velocity.
- Temperature:
  - Reaction velocity increases with temperature, up to a maximum point.
  - Beyond this point, enzyme denaturation leads to a decline in activity.
  - Optimal temperature for most enzymes is 35-40°C.
  - Temperature coefficient Q10 describes the change in enzyme velocity with a 10°C increase in temperature.
- pH:
  - Most enzymes have optimal activity between pH 6-8.
  - Balance between enzyme denaturation at extreme pH values and effects on the charged state of the enzyme/substrate.
  - Exceptions include pepsin (pH 1-2), acid phosphatase (pH 4-5), and alkaline phosphatase (pH 10-11).
- Activators:
  - Certain metal cations (Mn, Mg, Zn, Ca, Co, Cu, Na, K) act as activators.
  - Two categories:
    - Metal-activated enzymes: Metal ions are not tightly held by the enzyme and can be easily exchanged.
    - Metalloenzymes: The metal ion is tightly bound.

Vr-cFBPase: A Case Study

Vr-cFBPase (fructose-1,6-bisphosphatase) is a key enzyme in gluconeogenesis.
It is a 341 amino acid protein with 8 helices and 16 β-strands.
Vr-cFBPase has AMP and substrate (F16BP) binding domains identified.
Gene Cloning and Expression:
- The Vr-cFBPase gene was cloned from mung bean cDNA using PCR and TA cloning.
- The gene segment was inserted into a pET32a(+) expression vector.
- Site-directed mutagenesis was used to introduce mutations for further functional analysis.
- The gene was expressed in E. coli BL21(DE3) cells.
Recombinant protein characteristics:
- Vr-cFBPase exhibited a Michaelis-Menten constant (Km) of 7.96 μM.
- Turnover number (kcat) was 8.65 s-1.
- Catalytic efficiency (kcat/Km) was 1.09 μM s-1.
- The study observed an AMP inhibitory constant (Ki) of 111.09 μM.
- Met251 replacement with Leu (M251L) increased substrate affinity and activity.
- Asp32 was shown to be critical for maintaining the AMP binding conformation.
- Mutations in the active site affected binding energy and molecular distance.
- The Vr-cFBPase was found to be heat-sensitive.
Enzyme Immobilization:
- Enzyme immobilization is a technique used to confine enzyme molecules to a separate phase from the substrate and product.
- This improves enzyme stability and reduces cost by making the enzyme reusable.
- An enzyme can be immobilized by attaching it to an inert, insoluble material like calcium alginate.
- Immobilization enhances resistance to changes in pH, temperature, and other environmental factors.

Studying That Suits You

Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

Description

Explore the fascinating world of enzyme kinetics, which examines the rates of enzyme-catalyzed reactions and the factors that influence these rates. This summary discusses key concepts, including enzyme-substrate interactions and essential terms such as Km and Vmax. Gain insight into how substrate and enzyme concentrations affect reaction rates.