The document discusses enzyme kinetics and inhibition. It defines enzyme kinetics as the study of the rate at which enzymes catalyze biochemical reactions and how this rate changes with experimental parameters. It covers historical developments, factors affecting kinetics like substrate and enzyme concentration, temperature, and pH. It discusses the Michaelis-Menten equation and plot and the role of the enzyme-substrate complex. It also defines and describes different types of reversible and irreversible enzyme inhibition. Finally, it discusses the significance of enzyme kinetics in areas like metabolic regulation, cellular function, disease, drug design, and evolution.

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Advance Enzymology
Assignment No: 1
Topic: Exploring Enzyme kinetics and inhibition.
Submitted To: DR. Muhammad Akram
Submitted By: Faraz yaqoob
ID: F2023253014
MS( BC)

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Table of contents
1. **Enzyme Kinetics**
1.1 Definition
1.2 Historical Development
1.3 Adrina Brown's 1902 Study
2. **Factors Affecting Enzyme Kinetics**
2.1 Substrate Concentration
2.1.1 Michaelis-Menten Kinetics
2.2 Enzyme Concentration
2.3 Temperature
2.4 ph
3. **Role of ES Complex**
3.1 Michaelis and Menten's Contribution
3.2 ES Complex in Enzyme Kinetics
4. **Michaelis–Menten Equation and Plot**
4.1 Michaelis–Menten Equation
4.2 Michaelis–Menten Plot
5. **Enzyme Inhibition**
5.1 Definition
5.2 Types of Enzyme Inhibition

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5.2.1 Reversible Inhibition
5.2.1.1 Competitive Inhibition
5.2.1.2 Non-competitive Inhibition
5.2.1.3 Uncompetitive Inhibition
5.2.1.4 Mixed Inhibition
5.2.2 Irreversible Inhibition
6. **Biological Significances**
6.1 Metabolic Regulation
6.2 Cellular Function
6.3 Response to Environmental Changes
6.4 Disease and Therapy
6.5 Evolutionary Adaptation
7. **Drug Design**
7.1 Target Identification
7.2 Drug Screening and Development
7.3 Optimizing Drug Efficacy and Safety
7.4 Precision Medicine
7.5 Inhibitor Development
8. **Conclusion**
9. **References**

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Enzyme kinetics
Definition: Enzyme kinetics is the study of the rate at which enzyme catalyze any
biochemical reaction and also determine how it changes in response to changes in
experimental parameters.
History: The study of enzyme kinetics begin when in 1902, Adrina Brown reported
a study of rate of hydrolysis of sucrose which is catalyzed by yeast enzyme invertase.
The study shows that when sucrose concentration is much higher than that enzyme
than rate of reaction become independent of sucrose concentration.
(Segel, 1975).
Brown proposal: Adrina brown in 1902 proposed that overall an enzymatic
reaction is composed of two types of reaction in which substrate form a complex
with enzyme that gradually decompose to products and enzymes.
K1 K2
E+ S ES P + E
K2
Here, E, S, Es and P represents the enzyme, substrate, enzyme – substrate complex
and products.
According to model:
At high substrate concentration at which enzyme is completely converted into Es
complex, then 2nd
step become rate limiting step.

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The general expression of velocicity become:
𝑉 =
ⅆ(𝑝)
ⅆ𝑡
= 𝑘2 (𝐸𝑠)
(Zhang, Z. Y., & Wong, Y. N. (2020)
Factors affecting the Enzyme kinetics
1- Substrate concentration
Effect: According to Michaelis-Menten kinetics, the rate of enzyme reactions is
directly related to substrate concentration.
Reason: The saturation phenomenon is explained by the Michaelis-Menten
equation, which highlights the necessity of substrate availability for effective
enzyme activity.
2- Enzyme concentration :
Effect: Reaction rates are typically higher at higher enzyme concentrations.
Reason: The formation of the ES complex depends on the availability of the
enzyme, and an abundance of enzyme molecules increases the probability that
they will collide with substrates successfully.
3- Temperature:
Effect: Enzyme kinetics are affected by temp. Reaction rates normally rise up to
an ideal temperature, after which denaturation takes place.
Reason: Molecular mobility is enhanced by thermal energy, which raises the
possibility of efficient enzyme-substrate interactions. Yet the structural integrity
of the enzyme is compromised by high temperatures.
4- Ph
Effect: The optimum activity of enzymes is observed at particular pH values,
indicating the significance of the ionization state of amino acid residues.

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Reason: reason is because pH affects the charge on side chains of amino acids in
the active site, it has an impact on the attraction of substrates and the overall
structure of the enzyme. (Nelson & Cox, 2013).
Role of ES complex
The ES complex is the key to understanding the kinetic behavior of an enzyme.
In 1913, Leonor Michaelis and Maud Menten, developed a kinetic equation to
explain the behavior of many simple enzymes.
Key to the development of their equation, is the assumption that the enzyme first
combines with its substrate to form an enzyme-substrate complex in a relatively
fast reversible step:
k1
E + S ⇄ ES
K-1
Next, in a second, slower process, the ES complex decomposes to produce the
enzyme that is free and the reaction product P:
k2
ES ⇄ E + P
k-2
The total rate must be proportionate to the concentration of the species that reacts
during the second step, or ES, if the slower second reaction restricts the overall
reaction rate.
When [S] is high enough, almost all of the free enzyme has been changed to the
ES form, creating this situation.
The plateau seen in Fig. given below is caused by the saturation effect, which is
a distinctive feature of enzyme catalysts. Sometimes, the pattern depicted in such
image is called saturation kinetics. (Cornish-Bowden, 2012).

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Michaelis–Menten equation
Michaelis and Menten examined the invertase reaction`s starting rates at various
substrate concentrations in their article. They demonstrated that the initial rates of
the invertase reaction are accurately described by the Michaelis-Menten equation.
Equation:
(Cornish-Bowden, 2013)
Michaelis–Menten plot:
(Chou, T. C., & Talalay, P. (2018)

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Enzyme Inhibition
Definition: A substance that slows down or stops the normal catalytic function of
an enzyme by binding to the enzyme is called enzyme inhibitor.
OR
Enzyme inhibition refers to the process by which a molecule (inhibitor) modulates
or interferes with the activity of an enzyme, affecting the rate of an enzymatic
reaction. Morrison and Walsh (1988).
Types of Enzyme Inhibition
There are several types of enzyme inhibition, each with distinct mechanisms and
effects on enzyme activity:
1. Reversible Inhibition
● Competitive Inhibition
● Non-competitive Inhibition
● Uncompetitive Inhibition
● Mixed Inhibition
2. Irreversible Inhibition

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1. Reversible Inhibition
Inhibitors that can reversibly bind and dissociate from enzyme; activity of enzyme
recovers when inhibitor diluted out; usually non-covalent interaction.
(Stojan, J. 2005).
Competitive Inhibition
Mechanism:
Competitive inhibitors resemble the substrate and compete for the enzyme's active
site. When bound to the active site, they prevent the substrate from binding, reducing
the enzyme's ability to catalyze the reaction.
Effect:
Increasing substrate concentration can overcome competitive inhibition by
outcompeting the inhibitor for the active site.
Non-competitive Inhibition
Mechanism:
Non-competitive inhibitors bind to a site on the enzyme other than the active site,
known as the allosteric site. This binding causes a conformational change in the
enzyme's structure, reducing its catalytic activity.
Effect:
Non-competitive inhibition cannot be overcome by increasing substrate
concentration, as the inhibitor's binding site is distinct from the active site.

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Uncompetitive Inhibition
Mechanism:
Uncompetitive inhibitors bind only to the enzyme-substrate complex, forming an
enzyme-inhibitor-substrate complex. This binding reduces the enzyme's ability to
release products, slowing down the reaction rate.
Effect:
Uncompetitive inhibition is specific to the enzyme-substrate complex and affects
both substrate binding and product release (Segel, 1993).
Mixed Inhibition
Mechanism:
Mixed inhibitors bind to the enzyme or the enzyme-substrate complex at different
sites. They can either enhance or reduce enzyme activity, depending on their affinity
for the enzyme or the enzyme-substrate complex.
Effect:
Mixed inhibitors affect both the Km (Michaelis constant) and Vmax (maximum
velocity) of the enzymatic reaction (Dixon & Webb, 1979).

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2. Irreversible Inhibition:
Mechanism:
Irreversible inhibitors form strong covalent bonds or extensively bind to the enzyme,
rendering it permanently inactive. Also called suicide inhibition.
Effect:
Irreversible inhibition cannot be reversed, and new enzymes are needed for the
reaction to proceed.
Biological Significances
The biological significance of enzyme kinetics lies in its fundamental role in
governing the intricate biochemical processes essential for life:
1. Metabolic Regulation:
Enzyme kinetics governs metabolic pathways by regulating the rates of biochemical
reactions. Enzymes ensure that essential metabolic processes occur at the right rate
and time, maintaining cellular homeostasis (Berg, Tymoczko, & Gatto, 2019)
2. Cellular Function:
Enzymes drive cellular functions by catalyzing reactions involved in energy
production, DNA replication, protein synthesis, and cell signaling. Enzyme kinetics
determines the efficiency and specificity of these processes (Alberts et al. 2014).
3. Response to Environmental Changes: Enzymes adapt the cellular response to
environmental changes. Their kinetics allow cells to adjust their metabolic activities
based on external conditions, ensuring survival and adaptation (Feller, 2018).

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4. Disease and Therapy:
Understanding enzyme kinetics aids in comprehending diseases caused by enzyme
dysfunctions. This knowledge forms the basis for developing therapeutic
interventions that target enzymes, enabling the development of drugs to treat various
illnesses. (Walsh, 2005).
5. Evolutionary Adaptation:
Enzyme kinetics contributes to evolutionary adaptations. Changes in enzyme
kinetics over time can influence an organism's ability to adapt to new environmental
challenges, providing an advantage for survival. (Dean & Thornton 2007).
Enzyme kinetics, therefore, serves as the cornerstone of numerous biological
processes, influencing cellular function, adaptation, and disease mechanisms. Its
study is crucial not only in understanding the intricate workings of life but also in
advancing medical treatments, biotechnology, and sustainable practices
Drugs Design
Drug design refers to the process of discovering and creating new medications. It
involves identifying a specific target within the body related to a disease or
condition, finding or designing molecules that interact with this target, testing these
molecules for effectiveness and safety, and ultimately developing them into viable
medications. This process often combines scientific disciplines such as biology,
chemistry, pharmacology, and computational modeling to create drugs that
effectively treat diseases while minimizing side effects. (Silverman & Holladay 2018).
Enzyme kinetics plays a pivotal role in drug design by providing valuable
insights into the behavior of enzymes, enabling the development of medications that
specifically target these enzymes involved in diseases. Here's how:
1. Target Identification:
Enzyme kinetics helps identify crucial enzymes associated with diseases. By
understanding the kinetics of these enzymes, researchers can pinpoint which ones
play significant roles in disease progression. (Copeland, 2016).
2. Drug Screening and Development: Understanding enzyme kinetics aids in
screening and identifying molecules that interact selectively with these target
enzymes. This knowledge guides the design and optimization of drug compounds
that can modulate enzyme activity or inhibit specific enzymatic pathways.
(Segall 2010).

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3. Optimizing Drug Efficacy and Safety:
Enzyme kinetics assists in optimizing drug compounds to ensure effectiveness and
safety. By understanding how drugs interact with enzymes, researchers can modify
compounds to enhance their binding affinity, specificity, and pharmacokinetic
properties while minimizing adverse effects on healthy tissues (Di & Kerns 2015).
4. Precision Medicine:
Tailoring drugs to target specific enzymes involved in diseases enables more
targeted and personalized therapeutic interventions, enhancing the efficacy and
reducing side effects compared to broader-spectrum treatments. (Jameson & Longo
2018).
5. Inhibitor Development:
Enzyme kinetics studies various types of inhibition, aiding in the design of inhibitors
that can selectively target and modulate enzyme activity, thereby regulating disease-
related pathways. (Copeland, 2013).
Conclusion:
The study of enzyme kinetics is crucial for understanding the rate at which enzymes
catalyze biochemical reactions and how this rate is influenced by various
experimental parameters. The basis for later theories, such as Brown's theory of
enzymatic processes involving substrate-enzyme complexes, was established by
these early studies.
The saturation kinetics are further demonstrated by the Michaelis-Menten figure,
which highlights the significance of substrate concentration in enzyme processes.
Mathematical representations of enzyme behavior were supplied by the Michaelis-
Menten equation and plot, which also clarified the significance of the enzyme-
substrate (ES) combination in understanding enzyme kinetics.
The kinetic behavior of enzymes is mostly explained by the ES complex, with the
contributions of Leonor Michaelis and Maud Menten being especially noteworthy.
Enzyme kinetics is essential to drug design and is the basis for understanding a
variety of biological processes.
Researchers can create medications that specifically target the kinetics of the
enzymes linked to certain disorders, resulting in more individualized and efficient
treatment approaches.

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References :
1) Copeland, R. A. (2013). Enzymes: A Practical Introduction to Structure, Mechanism,
and Data Analysis (2nd ed.). Wiley.
2) Jameson, J. L., & Longo, D. L. (Eds.). (2018). Precision Medicine: A Guide to
Genomics in Clinical Practice. McGraw-Hill Education.
3) Di, L., & Kerns, E. H. (2015). Drug-Like Properties: Concepts, Structure Design and
Methods: From ADME to Toxicity Optimization (2nd ed.). Academic Press.
4) Segall, M. D. (2010). Drug-Like Properties: Concepts, Structure Design and Methods
from ADME to Toxicity Optimization. Academic Press.
5) Copeland, R. A. (2016). Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide
for Medicinal Chemists and Pharmacologists (2nd ed.). Wiley.
6) Silverman, R. B., & Holladay, M. W. (2018). The Organic Chemistry of Drug Design
and Drug Action (4th ed.). Academic Press.
7) Dean, A. M., & Thornton, J. W. (2007). Mechanistic approaches to the study of
evolution: The functional synthesis. Nature Reviews Genetics, 8(9), 675–688.
8) Walsh, C. T. (2005). Posttranslational Modification of Proteins: Expanding Nature's
Inventory. Roberts & Company Publishers.
9) Feller, G. (2018). Enzyme function at cold adapted conditions and its potential
applications. Essays in Biochemistry, 62(3), 429–441.
10) Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2014).
Molecular Biology of the Cell (6th ed.). Garland Science.

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11) Berg, J. M., Tymoczko, J. L., & Gatto, G. J. (2019). Biochemistry. W.H. Freeman.
12) Morrison, J. F., & Walsh, C. T. (1988). The behavior and significance of slow-binding
enzyme inhibitors. Advances in Enzymology and Related Areas of Molecular Biology,
61, 201–301.
13) I.H. Segel, Enzyme Kinetics, John Wiley & Sons, New York, 1993.
14) M. Dixon, E.C. Weeb, Enzymes, 3rd ed., Longman, London, 1979.
15) J. Stojan, Enzyme Inhibition, in: H.J. Smith, C. Simons (Eds.), Enzymes and Their
Inhibition, Drug Development, CRC Press, London, 2005, chapter 4, pp. 149-169.
16) Nelson, D. L., & Cox, M. M. Lehninger Principles of Biochemistry 6th
Edition (2013)
17) Segel, I. H. (1975). Enzyme kinetics: behavior and analysis of rapid
equilibrium and steady state enzyme systems.
18) Cornish-Bowden A (2012) Fundamentals of Enzyme Kinetics, 4th edn.
Wiley-VCH, Weinheim, Germany.
19) Cornish-Bowden A (2013) The origins of enzyme kinetics. FEBS Lett 587,
2725–2730
20) Chou, T. C., & Talalay, P. (1977). A simple generalized equation for the
analysis of multiple inhibitions of Michaelis-Menten kinetic
systems. Journal of Biological Chemistry, 252(18), 6438-6442.
21) Zhang, Z. Y., & Wong, Y. N. (2020). Enzyme kinetics for clinically
relevant CYP inhibition. Current drug metabolism, 6(3), 241-257.