CHM219-L7_74db0d70f1c8ea5d52a41de6c43b60e9
Page 1: Introduction
Course Title: Biochemistry I (CHM219)
Instructor: Assist. Prof. Dr. Esra Aydemir
Page 2: Topic Introduction
Focus on Enzymes II: The Biological Catalysts
Page 3: Outline of Key Topics
Kinetics of Enzymatic Catalysis
Enzyme Inhibition
Cofactors, Vitamins, and Essential Metals
Diversity of Enzymatic Function
Nonprotein Biocatalysts: Catalytic Nucleic Acids
Regulation of Enzyme Activity: Allosteric Enzymes
Covalent Modifications: Used to Regulate Enzyme Activity
Page 4: Conformational Selection vs. Induced Fit
Induced Fit Model: Enzyme is homogenous when unbound; changes upon substrate binding.
Conformational Selection Model: Enzyme exists in multiple conformations; substrate binds only to the specific conformation.
Le Chatelier’s Principle: Binding of substrate alters the equilibrium towards the ES complex.
Page 5: Kinetics of Enzymatic Catalysis
Expression for a reaction with a single substrate and product.
Assumption: k1, k-1, k3 >> k2 simplifies rate equations.
kcat: Apparent rate constant for substrate to product conversion.
Page 6: Reaction Rates and Steady-State
Initial conditions assumed to neglect reverse reaction between E and P.
Rate: First-order reaction dependent on [ES] and kcat.
Velocity expressed as: v = kcat[ES]
Difficulty in measuring [ES] during experiments.
Page 7: Steady-State Assumption
Desirable to express rate (v) in terms of [S] and total enzyme concentration.
Assumes equilibrium between E, S, and ES.
Steady-state conditions persist until substrate is almost consumed.
Page 8: Michaelis–Menten Equation
Rate equation relates initial reaction rate (v0), max rate (Vmax), and substrate concentration [S].
Michaelis Constant (KM): Measure of substrate-binding affinity.
Page 9: Reaction Velocity vs. Substrate Concentration
Graph of reaction velocity and [S] according to Michaelis–Menten model.
At [S] = KM, reaction rate is half of Vmax.
Vmax approached asymptotically.
Page 10: Steady-State Implications
Steady-state implies constant ES concentration.
KM indicates substrate concentration for half Vmax.
Turnover Number (kcat): Measures catalytic rate.
Page 11: Michaelis-Menten Parameters Table
Enzyme | Reaction Catalyzed | m (mol/L) | kcat (s-1) | Efficiency (kcat/KM) |
|---|---|---|---|---|
Chymotrypsin | Ac-Phe-Ala to Ac-Phe | 1.5 x 10-2 | 0.14 | 9.3 |
Pepsin | Phe-Gly to Phe | 3 x 10-4 | 0.5 | 1.7 x 10³ |
Tyrosyl-tRNA synthetase | Tyrosine + tRNA | 9 x 10-4 | 7.6 | 8.4 x 10³ |
Ribonuclease | Cytidine 2', 3' to | 7.9 x 10-3 | 7.9 x 10² | 1.0 x 10⁵ |
Carbonic anhydrase | Water | 2.6 x 10-2 | 1.5 x 10⁷ |
Page 12: Preferences of Chymotrypsin
Amino Acid | Side Chain | kcat/km |
|---|---|---|
Glycine | -H | 1.3 x 10-1 |
Norvaline | -CH2CH2CH3 | 3.6 x 10² |
Norleucine | -CH2CH2CH2CH3 | 3.0 x 10³ |
Phenylalanine | CH2 | 1.0 x 10⁵ |
Page 13: Analysis of Initial Rates
(a) Initial rates determined from varying substrate concentrations. (b) Plotted data shows adherence to Michaelis–Menten kinetics.
Page 14: Lineweaver–Burk Plot
Double reciprocal plot of 1/v vs. 1/[S] for determining Vmax and KM.
Prone to errors due to the nature of reciprocal measurements.
Page 15: Eadie–Hofstee Plot
Graph of v vs. V/[S], yielding Vmax at (V/[S]) = 0 and KM from slope.
Offers a graphical method for kinetic analysis.
Page 16: Kinetics Analysis Using Plots
Lineweaver–Burk and Eadie–Hofstee plots help determine KM and kcat.
Mutation effects analyzed through changes in KM and kcat values.
Page 17: Multisubstrate Reactions
Classes based on substrate binding order:
Random
Ordered
Ping-pong
Page 18: Random Substrate Binding
Either substrate can bind first, promoting the binding of the other substrate.
Page 19: Ordered Substrate Binding
One substrate must bind before the second can bind significantly.
Page 20: The Ping-Pong Mechanism
Cycle where one substrate binds, one product is released; then a second substrate and product.
Page 21: Rate Constants by Chymotrypsin
Table of rate constants for hydrolysis of N-acyl amino acid esters by chymotrypsin.
Page 22: Enzyme Inhibition Overview
Inhibition can be reversible or irreversible.
Competitive Inhibition: Inhibitor competes with substrate for the active site; increases apparent KM.
Page 23: Equilibrium and Substrate Interaction
The equilibrium state involves bound and free enzyme interactions with substrate and inhibitor.
Page 24: Competitive Inhibition Effects
Analysis of how competitive inhibitors affect reaction velocity at varying substrate concentrations.
Page 25: Lineweaver–Burk Plots of Inhibition
Competitive inhibitor lines intersect at 1/v demonstrating Vmax remains constant.
Page 26: KM and KI Determination
KI can be determined by varying concentrations of inhibitor, yielding KM when [I] = 0.
Page 27: Substrate and Inhibitor Examples
Example of UpA substrate competing with UpcA inhibitor for ribonuclease.
Page 28: Uncompetitive Inhibition Characteristics
Inhibitor does not compete for the active site, reducing Vmax and KM.
Page 29: Uncompetitive Inhibition Kinetics
Model displaying the interaction of E, S, and uncompetitive inhibition effects.
Page 30: Uncompetitive Inhibition Effects
(a) Effect of uncompetitive inhibitor on reaction velocity. (b) Parallel lines in Lineweaver–Burk plots indicate uncompetitive inhibition.
Page 31: Mixed Inhibition Model
Inhibitor binds to both free enzyme and ES complex, affecting substrate binding and catalytic events.
Page 32: Mixed Inhibition Mechanism
Kinetic model depicting the interaction of the enzyme with substrate and inhibitor.
Page 33: Lineweaver–Burk Plot for Mixed Inhibition
Vmax decreased; KM increased indicating mixed inhibition characteristics.
Page 34: Irreversible Inhibitors Overview
Irreversible inhibitors often bind covalently to enzymes; example: DFP.
Page 35: Irreversible Enzyme Inhibitors Table
Name | Formula | Source | Mode of Action |
|---|---|---|---|
Cyanide | CN- | Bitter almonds | Reacts with enzyme metal ions |
Diisopropyl F | Fluorophosphate | Synthetic | Inhibits serine-containing enzymes |
Sarin | F | Synthetic | Similar to DFP; a nerve gas |
Physostigmine | Calabar beans | Similar to DFP | |
Parathion | Synthetic | Inhibitor of acetylcholinesterase | |
Penicillin | ... | Penicillium fungus | Inhibitor of bacterial cell wall synthesis |
Page 36: Cofactors, Vitamins, and Essential Metals
Table of important enzyme cofactors and their associated vitamins.
Page 37: Nicotinamide Nucleotides
NAD+ as a key cofactor derived from niacin, crucial in redox reactions.
Page 38: NAD+ as Reducing Agent
NAD+ acts as an oxidizing agent in biological reactions, reversible electron transfer.
Page 39: Typical Reactions with NAD+
Role of NAD+ in converting alcohols to aldehydes or ketones via dehydrogenase.
Page 40: Trace Elements as Cofactors
Importance of trace metals like Fe, Cu, Zn in enzymatic functions.
Page 41: Diversity of Enzymatic Function
Some enzymatic reactions depend on metal ions for catalytic activity.
Page 42: Enzyme Classification by IUBMB
Six major enzyme classes defined by reaction types:
Oxidoreductases
Transferases
Hydrolases
Lyases
Isomerases
Ligases
Page 43: Examples of Major Enzyme Classes
Class | Example | Reaction Catalyzed |
|---|---|---|
Oxidoreductases | Alcohol dehydrogenase | CH3CH2OH to Acetaldehyde |
Transferases | Hexokinase | D-Glucose to D-Glucose-6-P |
Hydrolases | Carboxypeptidase A | Peptide bond hydrolysis |
Ligases | Pyruvate carboxylase | Carboxylation reaction |
Page 44: Nonprotein Biocatalysts
Ribozymes: RNA molecules capable of catalyzing specific reactions.
Page 45: DNAzymes and Rate Enhancements
Examples of reactions catalyzed by DNAzymes, with associated rate enhancements.
Page 46: Allosteric Enzymes
Regulation of enzyme activity crucial for metabolic pathways; feedback control.
Page 47: Cooperative Substrate Binding
Comparison of kinetic curves for noncooperative vs. allosteric enzymes.
Page 48: Extreme Homoallostery
Enzyme activity sharply increases at specific substrate concentration levels.
Page 49: Characteristics of Allosteric Enzymes
Display sigmoidal v vs. [S] curves; can be affected by various inhibitors and activators.
Page 50: Control Points in Pyrimidine Synthesis
Essential regulation points in the synthetic pathway, involving aspartate carbamoyltransferase.
Page 51: Regulation by ATP and CTP
ATP as an activator and CTP as an inhibitor of ATCase activity.
Page 52: Quaternary Structure of ATCase
Structure details and transitions involved between T and R states.
Page 53: Covalent Modifications Overview
Four types of covalent modifications that influence enzyme activities.
Page 54: Kinases and Phosphatases
Mechanisms by which kinases and phosphatases regulate enzyme activity via reversible modification.
Page 55: Proteolytic Cleavage for Activation
Certain enzymes activated irreversibly through cleaving zymogens.
Page 56: Activation of Chymotrypsinogen
Cleavage process yielding active chymotrypsin from its zymogen form.
Page 57: Blood Clotting Process
Cascade of proteolytic activations leading to fibrin formation.
Page 58: Formation of a Blood Clot
Detailed explanation of blood clot formation with fibrin monomer interaction.