Enzyme Kinetics Lecture Notes PDF

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

Dr Katja Vogt Enzyme kinetics @katjetz [email protected]  Dr Katja Vogt I will use the traffic light system through out my slides to help you evaluate the significance of the material @katjetz [email protected]  Dr Katja Vogt Today we are going to… … define enzymes as biological catalysts … explore different theories how enzymes function … identify the term and importance of enzyme kinetics … interpret the roles of isoenzymes … investigate the function of enzyme inhibition … highlight the regulation of enzyme activity @katjetz [email protected]  Dr Katja Vogt You have already come across several enzymes Lysosomal enzymes Polymerases Mitochondrial ATPase … @katjetz [email protected]  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]  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]  Dr Katja Vogt Bogumil Zelent et al. Biochem. J. 2008;413:269-280 @katjetz [email protected]  Dr Katja Vogt @katjetz [email protected]  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]  Dr Katja Vogt Classes of enzymes Class Type Action Examples of common names Dehydrogenase, oxidase, I Oxidoreductases Oxidation–reduction reactions peroxidase, reductase Transfer of amino, carboxyl, acyl, carbonyl, methyl, II Transferases Transaminase, transcarboxylase phosphate and other groups between molecules Esterase, peptidase, amylase, III Hydrolases Cleavage of bonds coupled with inserting water phosphatase, pepsin, trypsin Cleavage of carbon–carbon, carbon–sulphur and IV Lyases Decarboxylase, aldolase carbon–nitrogen (but not peptide) bonds V Isomerases Rearrangement of bonds Epimerase, mutase Formation of bonds between carbon and oxygen, VI Ligases Synthetase, carboxylase sulphur, nitrogen @katjetz [email protected]  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 Enzymes are the most efficient catalysts known @katjetz [email protected]  Dr Katja Vogt The Induced Fit Model vs Transition State Theory @katjetz [email protected]  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]  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 favor 3. strong binding 4. product released bind active site formation of transition state of transition state @katjetz [email protected]  Dr Katja Vogt Lysozyme is an example of Transition State 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]  Dr Katja Vogt Lysozyme is an example of Transition State Theory @katjetz [email protected]  Dr Katja Vogt The Induced Fit Model vs Transition State Theory @katjetz [email protected]  Dr Katja Vogt The Induced Fit Model E + S → ES Can → you E +think P of an example? Active site: pocket of amino-acids that bind substrate or play a role in catalysis Substrate binding distorts substrates into a conformation close to transition state Substrate binding can also induce conformation changes to the enzyme @katjetz [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 @katjetz [email protected]  Dr Katja Vogt Enzyme kinetics The rate of the reaction is affected by temperature, pH and substrate concentration @katjetz [email protected]  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]  Dr Katja Vogt Enzyme kinetics Saturation of enzyme with substrate (Vmax) Substrate concentration Vmax is dependent on the enzyme concentration @katjetz [email protected]  Dr Katja Vogt Saturation of enzyme Enzyme kinetics with substrate (Vmax) Km is equal to the substrate concentration at which the enzyme converts substrates into products at half its maximal rate Substrate concentration Km reflects the affinity of the substrate for the enzyme. @katjetz [email protected]  Dr Katja Vogt Compare and contrast the different curves @katjetz [email protected]  Dr Katja Vogt A and C à same enzyme concentration A and B à same affinity for substrate B à lowest enzyme concentration C à lowest affinity for substrate @katjetz [email protected]  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 initiated @katjetz [email protected]  Dr Katja Vogt Cytochrome P450 system Isoform Substrate Inducer Inhibitor Catalyse oxidation Tamoxifen Phenobarbital Orphenadrine CYP2B6 Cyclophospha Superfamily of microsomal mide Phenytoin (liver) enzymes Diazepam Phenobarbital Omeprazole Named as CYP +number + letter CYP2C8 Diclofenac Primidone Tolbutamide Paracetamol Ethanol Disulfiram Enflurane Isoniazid Ritonavir CYP2E1 Ethanol ! Isoenzyme vs Isoform ! Isoniazid @katjetz [email protected]  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]  Dr Katja Vogt Enzyme inhibition Non-competitive @katjetz [email protected]  Dr Katja Vogt Enzyme inhibition Competitive Non-competitive Inhibitor binds reversibly to Inhibitor bind outside of active active site site, decreasing the reaction rate Km increases Km remains unchanged Vmax remains unchanged Vmax decreases @katjetz [email protected]  Dr Katja Vogt Clinical relevance (example) Drugs may compete for the active site of a single cytochrome P450 enzyme le For examp Cimetidine (inhibits stomach acid production): bind competitively to several cytochrome P450 enzymes à reducing the metabolism of endogenous and administered steroids @katjetz [email protected]  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. Allosteric regulation @katjetz [email protected]  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]  Dr Katja Vogt eg. Phosphofructokinase Glyceraldehyde-3- P High [AMP] = low[ATP] phosphate P Glyceraldehyde-3- FRUCTOSE-6- 4 P phosphate P 2 PHOSPHATE 1 GLUCOSE-6- GLUCOSE PHOSPHATE ATP 3 Phosphofructokinase 1 ATP ADP 5 ADP P 4 FRUCTOSE-1,6- P KM P bisphosphate Dihydroxyacetone 4 phosphate @katjetz [email protected]  Dr Katja Vogt eg. Phosphofructokinase Glyceraldehyde-3- P High [AMP] = low[ATP] phosphate P Glyceraldehyde-3- FRUCTOSE-6- 4 P phosphate P 2 PHOSPHATE 1 What happens GLUCOSE-6- GLUCOSE PHOSPHATE to the Vmax in ATP 3 Phosphofructokinase 1 both reactions? ATP ADP 5 ADP P 4 FRUCTOSE-1,6- P P bisphosphate Dihydroxyacetone KM 4 phosphate @katjetz [email protected]  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 o r ex am p le F Chronic ethanol consumption induces a specific isoenzyme, CYP2E1 (substrates: ethanol, paracetamol, isoniazid) @katjetz [email protected]  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]  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 @katjetz [email protected]