4.2 Enzyme kinetics and inhibition
4 min read•august 1, 2024
Enzyme kinetics and inhibition are crucial for understanding how enzymes work in our bodies. This topic dives into the Michaelis-Menten model, which explains how enzymes interact with substrates and how fast reactions happen.
We'll explore different types of enzyme inhibitors and how they affect reaction speeds. We'll also learn how to measure and specificity, which are key to grasping how enzymes function in various biological processes.
Enzyme kinetics analysis
Michaelis-Menten model
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- Describes the kinetics of enzyme-catalyzed reactions
- Relates the initial reaction velocity (v0) to the [S]
- Assumes the enzyme (E) and substrate (S) form an enzyme-substrate complex (ES) in a reversible reaction
- ES complex then irreversibly breaks down to form the product (P) and regenerate the free enzyme
- Michaelis-Menten equation:
- represents the maximum reaction velocity when the enzyme is saturated with substrate
- Directly proportional to the total enzyme concentration [E]T
- is the Michaelis constant, the substrate concentration at which the reaction velocity is half of
- Represents the affinity of the enzyme for the substrate (lower indicates higher affinity)
- represents the maximum reaction velocity when the enzyme is saturated with substrate
Lineweaver-Burk plot
- Linear transformation of the Michaelis-Menten equation (double-reciprocal plot)
- Allows for the determination of and from experimental data
- Plots versus , yielding a straight line
- y-intercept of and an x-intercept of
- Used to analyze enzyme kinetics and determine kinetic parameters
- Helps visualize the effects of inhibitors on enzyme activity (changes in apparent and )
Inhibition effects on enzymes
Types of enzyme inhibitors
- Competitive inhibitors
- Bind reversibly to the of the enzyme, competing with the substrate
- Increase the apparent without affecting (higher substrate concentrations can outcompete the inhibitor)
- Examples: succinate dehydrogenase inhibited by malonate, dihydrofolate reductase inhibited by methotrexate
- Uncompetitive inhibitors
- Bind reversibly to the enzyme-substrate complex, not to the free enzyme
- Decrease both the apparent and (effectively remove ES complexes from the reaction)
- Examples: ATP synthase inhibited by oligomycin, acetylcholinesterase inhibited by physostigmine
- Mixed inhibitors
- Can bind to both the free enzyme and the enzyme-substrate complex
- Affect both and , with specific effects depending on relative affinities for E and ES
- Examples: aspartate transcarbamoylase inhibited by CTP, glycogen phosphorylase inhibited by glucose
Distinguishing inhibition types
- Lineweaver-Burk plots used to distinguish between inhibition types
- Patterns of changes in the apparent and indicate the type of inhibition
- : increased , unchanged (parallel lines with different x-intercepts)
- Uncompetitive inhibition: decreased and (parallel lines with different y-intercepts)
- Mixed inhibition: changes in both and (lines intersecting in the second or third quadrant)
Kinetic parameter determination
Experimental data analysis
- Measure initial reaction velocities (v0) at different substrate concentrations [S]
- Plot v0 versus [S] to obtain a hyperbolic curve (Michaelis-Menten plot)
- Use (double-reciprocal plot) to determine and
- Plot versus , yielding a straight line
- y-intercept of and an x-intercept of
- Calculate the () using the equation:
- is the total enzyme concentration
- represents the maximum number of substrate molecules converted to product per enzyme molecule per unit time
Enzyme efficiency and specificity constants
- Specificity constant () measures enzyme efficiency
- Reflects both the enzyme's affinity for the substrate () and its catalytic efficiency ()
- Higher indicates a more efficient enzyme
- Compare values for different substrates to assess enzyme specificity
- Enzymes with high specificity have a much higher for their preferred substrate compared to other potential substrates
- Use to evaluate the effectiveness of enzyme variants or mutants
- Mutations that improve enzyme efficiency or specificity will have increased values
Enzyme efficiency and specificity
Importance in biological processes
- Enzyme efficiency () is crucial for rapid and precise regulation of biochemical reactions
- Allows for maintenance of homeostasis and rapid response to changes in cellular conditions
- Enzymes can quickly catalyze reactions when needed
- Enzyme specificity ensures that enzymes catalyze only the desired reactions
- Prevents unwanted side reactions and maintains the fidelity of metabolic pathways
- Determined by the precise arrangement of amino acids in the active site, allowing for selective binding and catalysis of specific substrates
Consequences of altered efficiency or specificity
- Mutations affecting enzyme efficiency or specificity can lead to metabolic disorders and pathological conditions
- Highlight the importance of these properties in maintaining normal biological function
- Examples: phenylketonuria (mutation in phenylalanine hydroxylase), galactosemia (mutation in galactose-1-phosphate uridylyltransferase)
- Regulation of enzyme activity through various mechanisms allows for fine-tuning of metabolic pathways
- : binding of effectors at sites other than the active site modulates enzyme activity
- Post-translational modifications: phosphorylation, acetylation, or glycosylation can alter enzyme function
- Helps maintain homeostasis and adapt to changing cellular needs
Key Terms to Review (18)
Active Site: The active site is a specific region on an enzyme where substrate molecules bind and undergo a chemical reaction. This site is typically a pocket or groove on the enzyme's surface that is uniquely shaped to fit specific substrate molecules, facilitating the conversion of substrates into products while lowering the activation energy required for the reaction.
Allosteric regulation: Allosteric regulation refers to the modulation of an enzyme's activity through the binding of an effector molecule at a site other than the active site, known as the allosteric site. This process can either enhance or inhibit the enzyme's function, allowing for dynamic control of metabolic pathways and protein interactions. Allosteric regulation is crucial in various biological processes, as it influences enzyme kinetics, alters protein-ligand and protein-protein interactions, promotes cooperative binding among subunits, and is significant in single-molecule biophysics studies.
Amylase: Amylase is an enzyme that catalyzes the hydrolysis of starch into sugars, primarily maltose and dextrin. This enzyme plays a critical role in carbohydrate digestion, breaking down polysaccharides into simpler sugars, which can then be absorbed by the body. Amylase exists in different forms, mainly salivary amylase and pancreatic amylase, each with distinct functions in the digestive process.
Catalase: Catalase is an enzyme found in nearly all living organisms that catalyzes the decomposition of hydrogen peroxide into water and oxygen. This reaction is crucial as hydrogen peroxide is a harmful byproduct of many metabolic processes, and catalase helps to protect cells from oxidative damage by breaking it down efficiently.
Competitive Inhibition: Competitive inhibition is a type of enzyme inhibition where a molecule, known as an inhibitor, competes with the substrate for binding to the active site of an enzyme. This competition prevents the substrate from binding effectively, thereby reducing the rate of the reaction. Understanding competitive inhibition is crucial for studying how enzymes function and how their activities can be regulated in biochemical processes.
Covalent Modification: Covalent modification refers to the process of chemically altering a biomolecule, typically a protein or enzyme, through the formation of covalent bonds with other functional groups. This modification can significantly impact the activity, stability, and interactions of the biomolecule, playing a crucial role in regulating enzymatic functions and cellular processes.
Enzyme efficiency: Enzyme efficiency refers to the ability of an enzyme to catalyze reactions effectively, often measured by its turnover number (k_cat) and affinity for substrates (K_m). This concept is crucial for understanding how well enzymes perform under varying conditions and how they can be influenced by factors such as temperature, pH, and inhibitors. A higher enzyme efficiency indicates a more effective enzyme that can facilitate biochemical reactions at a faster rate.
Fluorescence Spectroscopy: Fluorescence spectroscopy is an analytical technique that measures the fluorescent light emitted by a sample after it has absorbed light or other electromagnetic radiation. This technique is widely used in various fields, including biology and chemistry, for understanding molecular interactions, studying enzyme kinetics, and investigating misfolding in proteins associated with diseases.
Hanes-Woolf Plot: The Hanes-Woolf plot is a graphical method used in enzyme kinetics to determine the kinetic parameters of an enzyme, particularly the Michaelis-Menten constant ($$K_m$$) and the maximum reaction velocity ($$V_{max}$$). This plot rearranges the Michaelis-Menten equation into a linear form, allowing for easier visualization and interpretation of enzyme activity and inhibition. By plotting $$ rac{[S]}{v}$$ (substrate concentration divided by reaction velocity) against $$[S]$$ (substrate concentration), it provides insights into how an enzyme's rate changes with varying substrate levels.
Km: Km, or the Michaelis constant, is a key parameter in enzyme kinetics that represents the substrate concentration at which the reaction rate is half of its maximum velocity (Vmax). This constant provides insight into the affinity of an enzyme for its substrate, with lower Km values indicating higher affinity and vice versa. Understanding Km is crucial for assessing enzyme efficiency and characterizing enzyme behavior in various biological processes.
Lineweaver-Burk Plot: The Lineweaver-Burk plot is a graphical representation used in enzyme kinetics to illustrate the relationship between enzyme activity and substrate concentration. It transforms the Michaelis-Menten equation into a linear format, allowing for the determination of key kinetic parameters like Vmax (maximum reaction rate) and Km (Michaelis constant) by plotting 1/V against 1/[S], where V is the reaction velocity and [S] is the substrate concentration. This plot is essential for understanding enzyme behavior and how different inhibitors can affect enzymatic activity.
Michaelis-Menten kinetics: Michaelis-Menten kinetics is a model that describes the rate of enzymatic reactions by relating reaction rate to substrate concentration. This model highlights the relationship between enzyme concentration, substrate saturation, and the resulting reaction velocity, making it essential for understanding enzyme behavior and mechanisms in biological systems.
Non-competitive inhibition: Non-competitive inhibition is a type of enzyme inhibition where an inhibitor binds to an enzyme at a site other than the active site, altering the enzyme's function regardless of the presence of the substrate. This results in a decrease in the overall rate of reaction because the inhibitor affects the enzyme's activity without competing with the substrate for the active site. This mechanism illustrates a key feature of enzyme kinetics, as it impacts how enzymes operate under various concentrations of substrate.
Specific Activity: Specific activity is a measure of enzyme activity that quantifies the amount of product formed per unit time per amount of enzyme present, usually expressed in units per milligram of protein. This term is crucial in understanding how efficiently an enzyme converts substrate into product under specific conditions and can vary based on factors such as enzyme concentration, substrate availability, and environmental conditions.
Spectrophotometry: Spectrophotometry is an analytical technique used to measure the amount of light that a sample absorbs at different wavelengths. This method is crucial for understanding various biochemical processes, as it allows researchers to quantify substances, analyze reaction rates, and study molecular interactions by monitoring changes in absorbance or fluorescence over time.
Substrate concentration: Substrate concentration refers to the amount of substrate present in a reaction mixture, which is crucial in determining the rate of enzymatic reactions. The level of substrate can significantly influence how quickly an enzyme catalyzes a reaction, as higher concentrations can lead to increased rates until saturation occurs. Understanding substrate concentration is essential for exploring enzyme kinetics and the effects of inhibitors on enzyme activity.
Turnover Number: Turnover number (often denoted as k\_cat) is a measure of the efficiency of an enzyme, defined as the number of substrate molecules converted to product by an enzyme molecule per unit time when the enzyme is fully saturated with substrate. This concept connects to reaction rates by illustrating how quickly a reaction can occur under optimal conditions and highlights the role of enzymes in speeding up reactions, offering insights into mechanisms of biochemical processes.
Vmax: Vmax is the maximum reaction rate achieved by an enzyme when it is saturated with substrate, meaning all active sites are occupied. This term is crucial in understanding how enzymes function, as it helps to characterize their efficiency and catalytic capabilities. The value of Vmax can vary between different enzymes and is influenced by factors such as enzyme concentration and temperature.