Page 1: Introduction to Enzymes

Enzymes

• Enzymes are biological catalysts that speed up biochemical reactions.

• They lower the activation energy required for reactions without being consumed in the process.

• They are essential for various metabolic pathways and are highly specific in their action.

Page 2: Enzyme Classification

TABLE 11-2: Enzyme Classification According to Reaction Classification

1. Oxidoreductases: Catalyze oxidation-reduction reactions.

2. Transferases: Involved in the transfer of functional groups.

3. Hydrolases: Catalyze hydrolysis reactions.

4. Lyases: Facilitate group elimination to form double bonds.

5. Isomerases: Catalyze isomerization processes.

6. Ligases: Form bonds, usually coupled with ATP hydrolysis.

Page 3: Definition of Enzymes

Characteristics of Enzymes

• An enzyme is either a protein or RNA molecule that catalyzes biochemical reactions.

• Enzymes bind specific substrates with high fidelity and specificity and enable reactions to occur under mild conditions.

• Most metabolic pathways are regulated through the regulation of enzyme functions.

Page 4: Enzyme Function (No Content)

Page 5: Specificity of Enzymes

Substrate Binding

• The active site of the enzyme is complementary to the substrate's structure, ensuring high specificity in substrate binding to initiate catalysis.

Page 6: Stereospecific Reactions

Catalysis of Stereospecific Reactions

• Enzymes like aconitase demonstrate high stereospecificity, where the binding and conversion of substrates yield specific stereoisomers (e.g., citrate to isocitrate).

Page 7: Geometric Specificity

Shape Dependency

• The geometric shape of the substrate is crucial for enzyme binding, exemplified by lysozyme binding to polysaccharides.

Page 8: Models of Enzyme-Substrate Interactions

Lock and Key Model

• The lock and key model illustrates how substrates bind to a pre-formed active site of the enzyme.

Page 9: Induced Fit Model

Induced Fit Mechanism

• The induced fit model describes how binding of substrates (e.g., by hexose kinase) causes conformational changes in the enzyme's structure, facilitating catalysis.

Page 10: Role of Catalysts

Comparison of Reaction Rates

• Example reaction: 2H2O2 → H2O + O2

  • No Catalyst: Activation Energy = 75.2 kJ mol-1, Relative Rate = 1

  • Platinum Surface Catalyst: Activation Energy = 48.9 kJ mol-1, Relative Rate = 2.77 x 104

  • Catalase Catalyst: Activation Energy = 23.0 kJ mol-1, Relative Rate = 6.51 x 108

Page 11: Mechanism of Catalysis

Reduction of Activation Energy

• Catalysts lower the activation energy (ΔG‡) of reactions while not affecting the change in free energy (ΔGo).

• Catalysts are not consumed during the reaction, allowing them to act repeatedly.

Page 12: Rate Acceleration Mechanisms

Methods to Accelerate Rates

• Increase local concentration of reactants.

• Increase concentration of reactive groups.

• Ensure proper orientation of reactants.

• Stabilize the transition state of the reaction.

Page 13: Example of Concentration Increase

Hexokinase Active Site

• Hexokinase provides an example where an increase in the concentration of reactants enhances reaction rates.

Page 14: Proximity Model

Support for the Proximity Model

• The kinetics of anhydride formation from esters and carboxylates show a strong dependence on the proximity of reactive groups.

Page 15: Reaction Rates and Base Catalysis

Peptide Hydrolysis Example

• Base catalyzed peptide hydrolysis is demonstrated showing the mechanism and involvement of reactive groups.

Page 16: Role of Cofactors

Cofactor Function

• Small molecules like NAD+/NADH, Biotin, and Riboflavin provide additional reactive groups for catalysis and are often obtained from the diet as vitamins.

Page 17: Lowering Activation Energy with Enzymes

Transition State Binding

• Enzyme active sites are often complementary to the transition state of the reaction, leading to stronger binding and lower activation barriers.

Page 18: Organizing Reactants

Enzyme Organization

• Enzymes organize reactive groups in close proximity to facilitate reactions, allowing favorable transition states to form.

Page 19: Enzyme Binding Energy

Rigid Reactant Complex Formation

• Enzymes use binding energy to create a rigid complex that reduces entropy issues encountered in uncatalyzed reactions.

Page 20: Free Energy Diagram Comparisons

Free Energy Changes

• Mappings of reaction states from substrate to product, showing the impacts of enzyme active site configurations on energy requirements.

Page 21: Transition State Stabilization

Supporting Data

• Transition state analogs demonstrate that stable structures bind more tightly than substrates, highlighting the potential for these analogs to act as inhibitors.

Page 22: Enzyme Assays

Measuring Reaction Rates

1. Color changes or UV absorbance.

2. Coupled assays that result in color changes.

3. Use of radioactive labels.

4. Experiments with colored substrate analogs.

Page 23: Initial Rate Measurements

Importance of Initial Rates

• Initial reaction rates are important for determining the kinetics of enzyme activities when only substrate concentration is considered influential.

Page 24: Analyzing Initial Rate Data

Graph Interpretation

• Analysis of graphs representing reaction rates at varying substrate concentrations aids in understanding enzyme kinetics.

Page 25: Michaelis-Menten Plot

Requirements for Analysis

• Each data point on the Michaelis-Menten plot corresponds to an experimental setup with a constant enzyme concentration and varying substrate concentrations.

Page 26: Michaelis-Menten Kinetics

Basic Equation Overview

• For the reaction S → P, if enzyme-catalyzed, the process involves the steps: E + S ↔ ES → E + P, resulting in rate constants k1, k-1, and k2.

Page 27: Rate-Determining Steps

Kinetics Breakdown

• In enzyme-catalyzed reactions, the rate is determined by the step involving the ES complex, mathematically represented as Rate = k2[ES].

Page 28: Equilibrium Approach 1

Assumption

• Assumes the first reaction reaches equilibrium, using equilibrium constants to derive rate equations for reaction mechanics.

Page 29: Steady State Assumption

Maintaining Stability

• The concentration of ES stays consistent, allowing for simplified calculations of rates based on enzyme and substrate concentrations.

Page 30: Michaelis-Menten Equation Derivation

Deriving Key Relationships

• Derivation shows that under steady-state conditions, the Michaelis-Menten equation can be simplified to rate = k2[E]t[S]/(Km + [S]).

Page 31: Final Form of Michaelis-Menten Equation

Rate Equation

• The equation derived shows how the maximum reaction rate (Vmax) and substrate concentrations impact the overall reaction velocity:

  • rate = k2[E][S]/(Km + [S]).

Page 32: Maximum Reaction Rates

Vmax Explanation

• Vmax represents the highest reaction rate when the enzyme is fully saturated with substrate, demonstrating first-order kinetics in enzymes.

Page 33: Km and Reaction Rate

Significance of Km

• At ranges where [S] = Km, the rate is half its maximum value, illustrating the relationship between substrate concentration and enzyme activity.

Page 34: Understanding Km

Km Details

• Km is crucial in determining how well substrates bind to enzymes and reflects both enzyme efficiency and interaction strength.

Page 35: Implications of Km Magnitude

Interpretation of Km Values

• A large Km indicates poor binding and fast conversion to products, while a small Km signifies strong binding and slower product formation.

Page 36: Normalizing Vmax

Vmax and Enzyme Concentration

• Normalizing Vmax to enzyme presence helps compare kinetic data across different enzymes, providing insight into enzyme efficiency.

Page 37: Clinical Relevance of kcat

Turnover Number

• kcat reflects the number of substrate molecules produced per enzyme per time unit, indicating catalytic efficiency but not directly related to Km.

Page 38: Catalytic Efficiency Measurement

Evaluating kcat/Km

• The kcat/Km ratio represents the catalytic efficiency, with values around 10^9 indicating high enzyme performance.

Page 39: Methods for Determining Vmax and Km

Kinetic Analysis Approaches

1. Direct fitting of data to the Michaelis-Menten equation.

2. Linearization methods such as Lineweaver-Burk and others for simplification.

Page 40: Lineweaver-Burk Plot

Simplification Method

• Rearranging the Michaelis-Menten equation into the Lineweaver-Burk format allows for linear analysis, facilitating estimation of Vmax and Km.

Page 41: Data Plotting Techniques

Visual Representation

• Importance of 1/[S] versus 1/V plots and how to derive rate constants from graphical representations.

Page 42: Analyzing Lineweaver-Burk Plots

Sensitivity to Error

• Discusses how minor rate errors can greatly impact parameter accuracy when using Lineweaver-Burk data.

Page 43: Alternative Kinetic Plots

Hanes Woolf and Their Pros/Cons

• Hanes Woolf plot advantages include less scatter, while its disadvantages include variable independence between axes.

Page 44: Eadie Hofstee Plot

Benefits and Drawbacks

• Provides direct readouts for Km and Vmax but suffers from non-independence of axes and requires careful interpretation.

Page 45: Direct Linear Plot Method

Median Values

• Discusses how direct linear methods provide median values for Vmax and Km through consistent plotting against various substrate concentrations.

Page 46: Best Methods for Kinetic Parameters

Effective Analytical Techniques

• Analyzing the best methods in calculating kinetic parameters and recognizing the utility of direct linear plotting for better data visualization.

Page 47: Significance of Enzyme Inhibitors

Enzyme Inhibition in Pharmaceuticals

• The importance of enzyme inhibitors in drug development, including therapeutic applications in HIV and anti-inflammatory medications.

Page 48: Types of Inhibition

Inhibition Mechanisms

• Competitive Inhibition: binds the active site in place of the substrate.

• Uncompetitive Inhibition: binds to another site only in presence of substrate.

• Mixed Inhibition: can bind either in presence/absence of substrate.

Page 49: Competitive Inhibition Details

Interactions and Impacts

• Competitive inhibitors can be outcompeted by high substrate concentrations, requiring comparison of binding constants to evaluate their effects.

Page 50: Curve Analysis in Competitive Inhibition

Graphical Representation

• Analyzing inhibitor effects by evaluating changes in Km with varying inhibitor concentrations and their impact on reaction kinetics.

Page 51: Uncompetitive Inhibition Explained

Mechanism of Action

• Uncompetitive inhibitors bind only to the ES complex, effectively reducing both Vmax and Km by altering enzyme conformation upon substrate binding.

Page 52: Lineweaver-Burk Analysis with Uncompetitive Inhibition

Implications for Kinetic Measurement

• Considers how uncompetitive inhibitors affect graphical display of Km and Vmax within the context of the Lineweaver-Burk plot.

Page 53: Mixed Inhibition Characteristics

Binding Behavior

• Mixed inhibitors can bind to free enzymes or the ES complex; non-competitive inhibition is a specific case.

Page 54: L-B Plot Implications

Kinetic Distinctiveness

• Kinetic variations like mixed inhibition can show intersecting lines below the x-axis within the Lineweaver-Burk format, indicating different inhibition effects.

Page 55: Effects of Inhibitors

Summary of Inhibition Effects

• Overview of how different types of inhibitors affect Michaelis-Menten equations, summarizing their impact on Vmax and Km.

Page 56: Irreversible Inhibitors

Mechanism of Action

• Irreversible inhibitors form stable covalent bonds with enzymes, illustrating their lasting effect on catalytic activity.

Page 57: Introduction to Bisubstrate Reactions

Two-Substrate Considerations

• Explains the approaches to measuring kinetic parameters when two substrates are involved in an enzymatic reaction.

Page 58: Bisubstrate Reaction Mechanisms

Types of Mechanisms

1. Ordered Binding: Specific sequential substrate binding yields a ternary complex.

2. Ping-Pong Mechanism: No ternary complex, summarized as enzyme transforming and releasing products consecutively.

Page 59: Data Plotting Techniques for Bisubstrate Reactions

Strategies for Analysis

• Utilizes excess substrate concentration in various scenarios to effectively measure and compareKm and Vmax values.

Page 60: Sequential Reactions

Enzyme-Catalyzed Pathways

• Flow of reactants through a series of stages to form end products, highlighting the complexity of kinetic analysis in sequences involving multiple substrates.

Page 61: Distinguishing Reaction Mechanisms

Product Inhibition Studies

• Use product inhibition as a method of differentiating between random and ordered mechanisms based on the behavior of inhibitors in enzyme pathways.

Page 62: Pointer on Kinetics

Ping-Pong Kinetics Description

• Summarizes how Ping-Pong kinetics operate in sequential reactions of enzymes, providing a succinct understanding of the mechanism.