Enzyme Kinetics Lecture Notes PDF

Dr Katja Vogt Enzyme kinetics [email protected] BSc Medical Sciences  Dr Katja Vogt I will use the traffic light system through out my slides to help you evaluate the significance of the material [email protected] BSc Medical Sciences ...

Dr Katja Vogt Enzyme kinetics [email protected] BSc Medical Sciences  Dr Katja Vogt I will use the traffic light system through out my slides to help you evaluate the significance of the material [email protected] BSc Medical Sciences  Dr Katja Vogt Dr Katja Vogt You have already come across several enzymes Lysosomal enzymes Polymerases Mitochondrial ATPase … @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Enzymes ⭐ Enzymes are proteins with unique structures that are folded to create 3-D active/catalytic sites which enable specific recognition of the substrates that they transform @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Enzymes ⭐ Enzymes are proteins with unique structures that are folded to create 3-D active/catalytic sites which enable specific recognition of the substrates that they transform @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt ⭐ Enzymes are proteins with unique structures that are folded to create 3-D active/catalytic @katjetz sites which enable specific recognition of the substrates that they [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Enzymes: Biological Catalysts Most biochemical reactions must be catalysed to proceed at rates that can support life Enzyme activity can be regulated and controlled according to the organism’s needs Enzymes are specific ENZYME SUBSTRATE PRODUCT @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Classes of enzymes Class Type Action Examples of common names Dehydrogenase, oxidase, I Oxidoreductases Oxidation–reduction reactions peroxidase, reductase II Transferases Transfer of amino, carboxyl, acyl, carbonyl, methyl, Transaminase, phosphate and other groups between molecules transcarboxylase Esterase, peptidase, amylase, III Hydrolases Cleavage of bonds coupled with inserting water phosphatase, pepsin, trypsin IV Lyases Cleavage of carbon–carbon, carbon–sulphur and Decarboxylase, aldolase carbon–nitrogen (but not peptide) bonds V Isomerases Rearrangement of bonds Epimerase, mutase Formation of bonds between carbon and oxygen, VI Ligases sulphur, nitrogen Synthetase, carboxylase @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Chemical reaction rates A catalyst increases the rate or velocity of a chemical reaction without being itself changed in the overall process Catalysts change the RATE not the EQUILIBRIUM of a reaction The equilibrium state is approached quicker using a catalyst Enzymes lower the activation energy For example: hydrogen peroxide on its own – breaks down into water and oxygen x1000 faster by adding iron (e.g. FeCl3) x million faster by adding haemoglobin (with iron in it) x billion faster by adding a catalase – hydrogen peroxidase @katjetz [email protected] Enzymes are the most efficient catalysts known [email protected]  Dr Katja Vogt Dr Katja Vogt @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt The Induced Fit Model vs Transition State Theory @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Transition states and reaction rates Enzymes reduce the activation energy of a reaction by binding to the transition structure and stabilising it E + S → ES → EP → E+P @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Transition states and reaction rates Transition state is stabilised by… 1. bind substrate(s) in the correct orientation 2. tightly binding transition state 1. substrates 2. proximity and orientation 3. strong 4. product bind active favor formation of transition binding of released @katjetz site state transition [email protected] state [email protected]  Dr Katja Vogt Dr Katja Vogt Lysozyme is an example of Transition Stat Theory Lysozyme breaks down peptidoglycan in bacterial cell walls. It is found in tears, saliva, human milk and mucus as part of body’s innate immune system. @katjetz [email protected] [email protected] Lysozyme is an example of TransitionDrState Dr Katja Vogt Katja Vogt Theory @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt The Induced Fit Model vs Transition State Theory @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt The Induced Fit Model E + S → ES → E +Substrate P binding distorts substrates into a conformation close to transition state Active site: pocket of amino-acids that bind substrate or play a role in catalysis Substrate binding can also induce conformation changes to the enzyme @katjetz [email protected] [email protected]  Dr Katja Vogt Hexokinase: Example of the Induced Fit Model Hexokinase –Binding of glucose causes 2 protein domains to close together – closing the binding cleft [email protected] BSc Medical Sciences  Dr Katja Vogt Dr Katja Vogt Enzyme kinetics @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Enzyme kinetics The rate of the reaction is affected by temperature, pH and substrate concentration @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Enzyme kinetics The rate of the reaction is reflected by the slope; the enzyme capacity is reflected by the hight of the curve Substrate concentration The rate of the reaction is affected by temperature, pH and substrate concentration @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Enzyme kinetics Saturation of enzyme with substrate (Vmax) Substrate concentration Vmax is dependent on the enzyme concentration @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Enzyme kinetics Saturation of enzyme with substrate (Vmax) Km is the Km is equal to the substrate substrate concentration at which the enzyme concentration at converts substrates into products at 2/Vmax Substrate concentration half its maximal rate Km reflects the affinity of the substrate for the enzyme. @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Compare and contrast the different curves @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt A and C  same enzyme concentration A and B  same affinity for substrate B  lowest enzyme concentration C  lowest affinity for substrate @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Isoenzymes ⭐ Enzymes that catalyse the same reaction. Can exist in different tissues in different forms Differ in substrate affinities and patterns of inhibition. Hexokinase 1 – in erythrocytes for fast energy production Glucokinase –in liver and pancreas where storage processes are @katjetz initiated [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Cytochrome P450 system Isoform Substrate Inducer Inhibitor Catalyse oxidation CYP2B6 Tamoxifen Phenobarbital Orphenadrine Cyclophosph Superfamily of amide Phenytoin Diazepam Phenobarbital Omeprazole microsomal (liver) CYP2C8 Diclofenac Primidone enzymes Tolbutamide Named as CYP +number Paracetamol Ethanol Disulfiram + letter CYP2E1 Enflurane Isoniazid Ritonavir Ethanol ! Isoenzyme vs Isoform ! Isoniazid @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Practical Units of Enzyme Measurement Activity of an enzyme = ‘specific activity’ μmol / min / mg Micromoles of substrate converted to product per minute per milligram of enzyme protein International Unit (U) / ml 1U = the quantity of enzyme which catalyses the conversion of 1μmol of substrate to product per minute under a defined set of optimal conditions katal (kat) Amount of enzyme that converts one mole of substrate to product per second @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Enzyme inhibition Non-competitive @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Enzyme inhibition Competitive Non-competitive Inhibitor binds reversibly to Inhibitor bind outside of active active site site, decreasing the reaction Km increases rate Vmax remains unchanged Km remains unchanged Vmax decreases @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Clinical relevance (example) Drugs may compete for the active site of a single cytochrome P450 enzyme Especially relevant for patients receiving multiple drugs r ex a mple Fo Cimetidine (inhibits stomach acid production): – bind competitively to several cytochrome P450 enzymes  reducing the metabolism of endogenous and administered steroids @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Allosteric regulation Usually multi-subunit proteins with multiple active sites Adapted from Fig 7.7 Mark’s Essentials of Medical Biochemistry @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt eg. Phosphofructokinase Glyceraldehyde- P 3-phosphate High [AMP] = low[ATP] P FRUCTOSE Glyceraldehyde- -6- 4 P 3-phosphate P 2 PHOSPHAT GLUCOSE E 1 GLUCO -6- SE PHOSPHA AT TE P 3 Phosphofructokinas AT AD e1 P 5 P AD P P 4 FRUCTOSE- 1,6- P P bisphosphate Dihydroxyacetone 4 phosphate @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt eg. Phosphofructokinase Glyceraldehyde- P 3-phosphate High [AMP] = low[ATP] P FRUCTOSE Glyceraldehyde- -6- 4 P 3-phosphate P 2 PHOSPHAT 1 GLUCOSE E What GLUCO -6- SE PHOSPHA AT happens to AT TE P 3 Phosphofructokinas the Vmax in AD e1 P P AD 5 both P reactions? P 4 FRUCTOSE- 1,6- P P bisphosphate Dihydroxyacetone KM 4 phosphate @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Non-competitive vs allosteric Changes the ability to Changes the affinity to carry on with the the substrate reaction Vmax, and Km can be Vmax, but not Km is changed changed Negative or positive Inhibitory only @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Why do we need to know this? Concepts can be applied to receptor - ligand binding too, and crucial concepts of pharmacology are overlapping here Receptors have an active binding site for their activating ligands Binding sites are highly specific Ligand binding induces conformational changes to activate receptor May also contain secondary In(“allosteric”) binding sites for other ligands Pharmacology : Ligand = @katjetz [email protected] [email protected] agonist  Dr Katja Vogt Affinity vs efficacy The ability of a ligand to The ability of a ligand to bind to a target generate an effect Substrate a has the same efficacy as substrate c, but has a lower affinity Substrate b has the same affinity as substrate a, but b has a lower efficacy [email protected] BSc Medical Sciences  Dr Katja Vogt Dr Katja Vogt Regulation of enzyme activity 1. Feedback loops 2. Feedforward activation 3. Phosphorylation–dephosphorylation sequence 4. Proteolysis 5. Changes in gene expression 6. Unspecific regulation 7. Allosteric regulation 8. Enzyme induction @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Enzyme induction Increased de novo synthesis of enzymes Mostly done by drugs, over a period of time (weeks) Slowly reversible Mechanism of induction is not fully understood ex a mpl e For Chronic ethanol consumption induces a specific isoenzyme, CYP2E1 (substrates: ethanol, paracetamol, isoniazid) @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt Can you… … define enzymes as biological catalysts? … describe different theories how enzymes function? … identify the term and importance of enzyme kinetics? … interpret the roles of isoenzymes? … illustrate the function of enzyme inhibition? … highlight the regulation of enzyme activity? MBBS learning outcomes Define enzyme structure and function including enzyme kinetics Define energy metabolism and carbohydrate biochemistry Recognise the application of biochemical profiling and their principles or mode of action @katjetz [email protected] [email protected]  Dr Katja Vogt Dr Katja Vogt References / further reading “Essentials of Medical Biochemistry” N.V.Bhagavan, Chung-Eun Ha. Chapter 6 “Medical Biochemistry at a Glance” S. G. Salway. 3rd edition. “Mark’s Basic Biochemistry” Lieberman and Marks. 4th edition “The Molecular Biology of the Cell” Alberts and Johnson. 6th edition. Chapter 3 https://www.youtube.com/watch?v=q94TCTSXyv8 @katjetz [email protected] [email protected]