Protein Function 01 - Enzymes and Regulation PDF

XY1110: BSc Medical Sciences Protein Function Enzymes and Regulation SGM104 Gillian Lewis [email protected]  Enzymes Learning objectives Define enzymes and understand how their structure relates to th...

XY1110: BSc Medical Sciences Protein Function Enzymes and Regulation SGM104 Gillian Lewis [email protected]  Enzymes Learning objectives Define enzymes and understand how their structure relates to their function Understand Michaelis-Menten enzyme kinetics Describe the features and extract the characteristics of enzymes from enzyme- saturation curves and Lineweaver-Burk plots  Enzymes Lecture Layout Amino acid and protein recap Common Features of Enzymes - Active site - Substrate docking - Activation energy - Temperature and pH Types of Catalysis Reaction Kinetics - Michaelis Menten Kinetics - Enzyme Saturation Curve - Michaelis Menten Equation - Lineweaver-Burk plot  Enzymes Amino Acids – Starter recap Proteins are made up of amino acids  Amino Acids - Recap 20 Different amino acids  Metabolism Enzymes Almost all reactions in living Biological Catalysts organisms (metabolism) are catalysed by enzymes Increase the rate of Metabolic reactions Anabolic reactions Globular Proteins, nucleic acids Catabolic reactions Active Site Substrate is turned into a Product.  Enzymes Substrate Product Often feature - Co-factors - Inorganic molecules (Fe2+, Zn2+, Mg2+, Cu2+, Mg2+, and Mn2+) Molecular Biology of the Cell  Enzymes Active site - Substrate docking models 1) Lock-and-key Model - Complementary 3D structures - No major atomic rearrangements 2) Induced fit model - Substrate induces a conformational change - substrate fits well after reshape Physical Chemistry 9th ed  Enzymes Active site - Structure Lysozyme (From PDB 6LYZ) - Active sites may include distant residues  Enzymes Catalysis - Activation Energy Activation energy Extra energy required to reach a transition state for a reaction to proceed. Catalyst - Lower the activation energy of a reaction → Increasing reaction rate - Not consumed by the reaction Molecular Biology of the Cell  Enzymes Catalysis - Optimal Conditions Temperature Activity accelerated by increased temperature High temperature → Enzyme denatures pH Protonation state of side groups - Enzyme and substrate Note: Blood pH 7.4 +/- 0.5 pH units → fatal pH range within cells pH 6.5-7.0 in cytoplasm pH 7.5-8.0 in the mitochondrial matrix pH 4.5 to 5.5 in the lysosomes Principles of Medical Biochemistry 4th Ed (2017) Catalysis - Optimal Conditions Various Conditions Temperature pH Ionic strength Substrate concentration Etc.  Enzymes Conformational Changes Hexokinase Enzymes Require Movement Glucose → glucose-6-phosphate Restrict movement → Decrease activity Change substrate environment - Favour product formation - Favour product release Open Closed (PDB entry 2yhx and 1hkg)  Enzymes Types of Catalysis 1) Acid-base catalysis - Enzyme acts as partial proton donor (acid) or acceptor (base) Example: Myosin - Motor protein Kodera et al. (2010) Nature 468:43 Kiani & Fischer (2016) Physical Chemistry Chemical Physics 18:20219  Enzymes Types of Catalysis 1) Acid-base catalysis 2) Covalent catalysis - Active site temporarily covalently modified Example: Chymotrypsin - Protease (C-side of aromatic or large hydrophobic amino acids) - Nucleophilic Serine Biochemistry 9th Edition (2019)  Enzymes Types of Catalysis 1) Acid-base catalysis 2) Covalent catalysis 3) Metal-ion catalysis i) Electrophilic catalyst - stabilising a negative charge ii) Generate a nucleophile (OH-) Example: Carbonic anhydrase - increasing the acidity of a nearby molecule H2O + CO2 → HCO3- + H+ - Zink ion - Reacts with water iii) Metal ion may bind to substrate - Acidic –OH2 Biochemistry 9th Edition (2019)  Enzymes Types of Catalysis 1) Acid-base catalysis NMP kinase 2) Covalent catalysis 3) Metal-ion catalysis 4) Approximation catalysis - Bringing two substrates together Biochemistry 9th Edition (2019)  Enzymes Cofactors - Definitions Cofactors Molecules that aid enzymatic function Note: Add variation to the 20 amino acids 1) Metal 2) Coenzymes Small organic cofactors A) Prosthetic groups Tightly bound coenzymes B) Cosubstrates Loosely associated coenzymes Biochemistry 9th Edition (2019)  Enzymes Reaction Kinetics Rate of a reaction (V) – Units of M s-1 Rate = Decrease in substrate over time Rate = Increase in product over time OR Biochemistry 9th Edition (2019)  Enzymes Reaction Kinetics Rate constant (k) - relates chemical concentrations to rate (V) First-order rate constants Second-order rate constants k has units of s-1 k has units of M-1s-1 Note: Pseudo-first-order reactions - e.g. when B is in excess Biochemistry 9th Edition (2019)  Enzymes Enzyme Kinetics - Michaelis Menten Kinetics Calculating the rate of a reaction (V) and rate constants (k) Substrate Product  Enzymes Enzyme Kinetics - Michaelis Menten Kinetics Calculating the rate of a reaction (V) and rate constants (k) Initial velocity (V0) Reaction rate at T0 No products Biochemistry 9th Edition (2019)  Enzymes Enzyme Kinetics - Michaelis Menten Kinetics Calculating the rate of a reaction (V) and rate constants (k) Enzyme Saturation Curve Biochemistry 9th Edition (2019)  Enzymes Enzyme Kinetics - Michaelis Menten Kinetics Calculating the rate of a reaction (V) and rate constants (k) Enzyme Saturation Curve Maximal velocity (Vmax) Reaction rate when enzyme is saturated All active sites filled Michaelis constant (KM) [Substrate] at half Vmax Biochemistry 9th Edition (2019)  Enzymes Enzyme Kinetics - Michaelis Menten Kinetics In steady state [ES] remains the same ES Formation = Breakdown  Enzymes Enzyme Kinetics - Michaelis Menten Kinetics In steady state Michaelis Constant Michaelis Menten Equation  Enzymes Enzyme Kinetics - Michaelis Menten Equation Enzyme Saturation Curve Michaelis Menten Equation 1) Low [S] First order reaction → V0 ∝ [S] 2) High [S] Zero order → V0 ≈ VMAX 3) [S] = KM V0 = VMAX/2 Biochemistry 9th Edition (2019)  Enzymes Enzyme Kinetics - Michaelis Menten Equation Enzyme Saturation Curve VMAX Changes in Enzyme saturation curve 1) Downward shift in curve Decrease VMAX 2) Upward shift in curve Increase VMAX VMAX 3) Right Shift in curve Increase KM (lower binding) 4) Left Shift curve Decrease KM KM KM (Higher binding) Biochemistry 9th Edition (2019)  Enzymes Hexokinase and Glucokinase - Michaelis Menten Kinetics Initial step in Glycolysis Hexokinase or Glucokinase Glucokinase (Liver and in the pancreatic β-cell) High VMAX High KM → Respond to large [Glucose] - [Glucose] sensor Hexokinase Lower VMAX Lower KM Not specific to glucose  Enzymes Enzyme Kinetics - Michaelis Menten Kinetics Enzyme Saturation Curve Lineweaver-Burk plot y = m𝑥 + c Biochemistry 9th Edition (2019)  Enzymes Enzyme Kinetics - Variables [Enzyme] Lineweaver-Burk plot VMAX depends on enzyme concentration Doubling [enzyme] → double reaction rate Increase [Enzyme]  Enzymes Summary Common Features of Enzymes Reaction Kinetics - Active site - Michaelis Menten Kinetics - Substrate docking - induced fit - Enzyme Saturation Curve - Activation energy - lower - Michaelis Menten Equation - Lineweaver-Burk plot Types of Catalysis - Temperature and pH 1) Acid-base catalysis (H+ Donor/Acceptors) 2) Covalent catalysis 3) Metal-ion catalysis 4) Approximation catalysis y = m𝑥 + c  Enzymes Recommended reading Biochemistry Principles of Medical 9th Edition Biochemistry Chapters 8 & 9 4th Edition Chapters 4 & 5  Enzyme Regulation Learning objectives Understand how enzymatic reactions can be regulated Describe the features of the different types of enzymatic inhibition  Enzyme Regulation Lecture Layout Metabolic Pathway Steps Enzymatic Inhibition Enzymatic Regulation  Enzyme Regulation Metabolic Pathways Nomenclature Rate-limiting Step - Slowest step in a pathway Rate-limiting & - Highest activation energy Committed step - Limited by [S] Committed step - First irreversible enzymatic reaction in a pathway to a specific product. 3 4 1 2 C D E A B 5 F G H 6 7 Moreno-Sanchez et al (2008) Journal of Biomedicine and Biotechnology 7:597913  Enzyme Regulation Types of Regulation 1) Allosteric 2) Multiple Forms of Enzymes (isozymes) 3) Reversible Covalent Modification 4) Proteolytic Activation 5) Expression control 6) Inhibition  Enzyme Regulation Inhibitors - Type of Inhibition 1) Competitive 2) Uncompetitive 3) Non-competitive -  Enzyme Regulation Inhibitors - Type of Inhibition 4) Irreversible/suicide inhibitors - Inhibitor covalently binds enzyme - Reacts with active site Example : Penicillin inhibition of DD-transpeptidase cell wall synthesis 5) Product inhibition - Product negative feedback  Enzymes Recap: Enzyme Kinetics - Michaelis Menten Kinetics Enzyme Saturation Curve Lineweaver-Burk plot y = m𝑥 + c Biochemistry 9th Edition (2019)  Enzyme Regulation Inhibitors - On reaction VMAX and KM 1) Competitive - VMAX remains the same - KM increases (More [S] needed) - Slope is steeper (KM/VMAX) Lineweaver-Burk plot Enzyme Saturation Curve Biochemistry 9th Edition (2019)  Enzyme Regulation Inhibitors - On reaction VMAX and KM 2) Uncompetitive - VMAX is lower - KM is lower (More ES complexes) - No change in slope (ΔVMAX ∝ ΔKM) Lineweaver-Burk plot Enzyme Saturation Curve Biochemistry 9th Edition (2019)  Enzyme Regulation Inhibitors - On reaction VMAX and KM 3) Non-competitive - VMAX is lower - KM remains the same - Slope is steeper Enzyme Saturation Curve Lineweaver-Burk plot Biochemistry 9th Edition (2019)  Enzyme Regulation Allosteric Regulation - Cooperative Effects Allosteric Regulation Activity at one functional site affects the activity at others. Allosteric proteins contain 1) Distinct regulatory sites 2) Multiple functional sites Concerted model Biochemistry 9th Edition (2019)  Enzyme Regulation Allosteric Regulation - Cooperative Effects Allosteric Regulation Biochemistry 9th Edition (2019)  Enzyme Regulation Allosteric Regulation - Enzyme-saturation curve Enzyme-saturation curve Simple Sigmoidal curve (S-shape) reaction - Velocity increases over a narrower range - Allows for “on/off” switch Allosteric Regulation Biochemistry 9th Edition (2019)  Biochemistry 9th Edition (2019) Enzyme Regulation Types of Regulation - Covalent Modifications Reversible Covalent Modification Attachment of modifying group.  Enzyme Regulation Types of Regulation - Zymogens/proenzymes Zymogens/proenzymes Enzymes that need activating by another enzyme to function. Examples: Pepsin Activation (Stomach) Collagen Synthesis Pepsinogen H+ Active Pepsinogen Secreated Procollagen Peptide Cleavage Pepsin Collagen Molecular Biology of Collagen Fibril the Cell 6th Edition  Enzyme Regulation Types of Regulation - Zymogens/proenzymes Zymogens/proenzymes Enzymes that need activating by another enzyme to function. Examples: Caspases in Apoptosis Molecular Biology of the Cell 6th Edition  Enzyme Regulation Summary Metabolic Pathway Steps Enzymatic Regulation - Rate-limiting Step 1) Allosteric - Committed step 2) Multiple Forms of Enzymes (isozymes) 3) Reversible Covalent Modification Enzymatic Inhibition 4) Proteolytic Activation 1) Competitive 5) Expression control 2) Uncompetitive 6) Inhibition 3) Non-competitive 4) Irreversible/suicide inhibitors 5) Product inhibition  Enzyme Regulation Recommended reading Biochemistry 9th Edition Chapter 10