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Questions and Answers
What happens to the rate of an enzyme reaction as the temperature increases beyond its optimum level?
How does the concentration of enzyme affect the reaction velocity?
What is the optimum pH range for most enzymes?
Which of the following statements about activators is true?
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What characterizes a metalloenzyme?
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What does the rate law express in relation to chemical reactions?
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What is the primary focus of enzyme kinetics?
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How do enzyme-catalyzed reactions behave as substrate concentration increases?
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What is the initial observation made by Adrina Brown regarding enzyme kinetics?
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Which terms are commonly used to model catalytic efficiency in enzyme kinetics?
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What type of reactions does the rate law apply to?
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What characterizes a first-order reaction in the context of the rate law?
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What does the term 'enzyme-substrate complex' refer to?
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What is the predicted size of the PCR product from the TA cloning of the VrFBP gene?
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Which polymerase is used for the PCR in the TA cloning of VrFBP gene?
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What process follows the ligation in the construction of the recombinant plasmid?
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What is the role of T4 ligase in recombinant plasmid construction?
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What is the purpose of Dpn I digestion in the plasmid construction process?
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What type of chromatography is used for protein purification in the described process?
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What is the composition of Vr-cFBPase according to the provided information?
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During the TA cloning process, which selection method is employed to identify successful clones?
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What is the significance of A-tailing in the PCR process?
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What type of cell is indicated for transformation in the process described?
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What initial step is taken to create a cDNA library from mung bean mRNA?
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Which type of cloning is indicated for the VrFBP gene in the content provided?
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What induces protein expression in the PET32-FBP vector system?
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Which component of the protein structure allows for specific interactions with substrates?
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What effect did the replacement of Met251 by Leu have on enzyme performance?
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Which amino acid is crucial for maintaining the AMP binding conformation?
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What is the primary challenge associated with the use of soluble enzymes in industrial applications?
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What defines an immobilized enzyme?
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What is a benefit of enzyme immobilization?
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Which characteristic of enzymes limits their widespread use in industries?
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What process is described as confining enzyme molecules to a distinct phase from the substrates and products?
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What environmental factors can immobilized enzymes provide increased resistance to?
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What is the purpose of the Nde I and BamH I restriction enzymes in the construction of the PET32-FBP plasmid?
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What is the consequence of performing site-directed mutagenesis on the PET32-FBP plasmid?
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What is the primary role of T4 ligase in plasmid construction?
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Which of the following represents the catalytic efficiency of the enzyme as defined by Kcat/Km?
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What does the term 'turnover number' (Kcat) refer to?
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What effect does the AMP inhibitory constant (Ki) represent regarding enzyme activity?
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Which step is crucial for verifying the successful transformation of the PET32-FBP-DH5α plasmid?
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What is the significance of using methylated plasmid in site-directed mutagenesis?
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What does the term 'temperature sensitive' imply regarding the enzyme used in the experiments?
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What is the predicted size of the product in the colony PCR?
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Which of the following would NOT be a step in the protein expression process outlined?
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What does the presence of a supercoil in plasmid DNA indicate?
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What is the role of ion exchange chromatography in the purification of proteins?
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What type of protein is most likely being expressed in the PET32-FBP plasmid system?
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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.
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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.
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Factors affecting enzyme reaction rate:
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Substrate concentration:
- Higher concentration leads to more collisions and a faster reaction rate.
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Enzyme concentration:
- Higher enzyme concentration leads to a directly proportional increase in reaction velocity.
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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.
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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).
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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.
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Substrate concentration:
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.
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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.
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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.
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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.
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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.