BIO211 Lecture Notes - Enzymes I - Properties and Kinetics - PDF

28/03/2023 Enzymes The catalyst of life 1 Enzymes are Biological Catalysts Enzyme are proteins which have to unique properties: 1. They are powerful catalysts, which accelerate...

28/03/2023 Enzymes The catalyst of life 1 Enzymes are Biological Catalysts Enzyme are proteins which have to unique properties: 1. They are powerful catalysts, which accelerate biochemical reactions, sometimes millionfold 2. They are very reaction specific, catalysing a single reaction or a set of closely related reactions 2 1  28/03/2023 Enzymes are Biological Catalysts Are globular proteins. Specific 3D shape Speed up reactions by lowering activation. Do not get used up during the rxn. Are specific to reactions they catalyse. Active site Have optimal environmental conditions. temp. pH, [salt] 3 3 Enzymes are Biological Catalysts Carbonic anhydrase Enzyme (E) CO2 + H 2O H2CO3 Substrates (S) Products (P) Rate (molecules/s) Uncatalysed 0.13 Catalysed 1,000,000 Enzymes accelerate reactions by factors of as much as a million or more. 4 2  28/03/2023 Enzymes are Biological Catalysts 5 The Active Site Cluster of amino acids - substrate binds Takes up a relatively small part of total volume of enzyme (few amino acids) Usually groove or pocket → microenvironment 6 3  28/03/2023 The Active Sites of Enzymes have some Common Features Active site: region in enzyme that binds substrates Binding groups: temporarily binds to substrate Catalytic groups: residues that participate in bond- formation or breaking Lysozyme - Peptidoglycan 7 8 4  28/03/2023 Complementarity of Enzyme to Substrate Specificity: refers to the ability of the protein to bind one molecule in preference to others. Affinity: refers to the strength of binding 9 Enzymes are highly specific Endopeptidase Gly His Phe Gly Try Lys Arg Trp Gly Pepsin Gly His Phe Gly Try Lys Arg Trp Gly Chymotrypsin Gly His Phe Gly Try Lys Arg Trp Gly Trypsin Exopeptidase Gly His Phe Gly Try Lys Arg Trp Gly Aminopeptidase Gly His Phe Gly Try Lys Arg Trp Gly Caboxypeptidase 10 5  28/03/2023 Enzymes Sensitivity Enzymes are most effective at optimal conditions 11 Different Enzymes have different Sensitivities 12 6  28/03/2023 The Catalytic Event Enzymes are not changed or used up 13 Enzymes ONLY Changes the Rate at which a Reaction Occurs 14 7  28/03/2023 Enzymes Reduce the Energy Needed to Initiate Spontaneous Reactions S+E ES P+E Facilitate the formation of a Transition state – Enzyme substrate complex 15 16 Enzymes Kinetics Studies of enzyme rates with changes in substrate[S], product [P] and inhibitor [I] determine to how enzymes work. Why analyze the kinetics of enzymes? Important in the discovery of new drugs Industrial synthesis of chemicals Understanding the chemistry of cells in living organisms 16 8  28/03/2023 17 Michaelis-Menten kinetics Vmax Reaction Rate (Vo) + Michaelis – Menten equation Substrate Concentration [S] 17 18 Michaelis-Menten kinetics Initial reaction velocity (V0) Plot of product formed [P] versus time S+E ES P+E Enzyme Product [P] Slope = V0 Substrate Time 18 9  28/03/2023 Michaelis-Menten kinetics Relationship between (V0) and [S] Carry out series of rxns with varying [S] and determine initial velocity (V0) for each reaction 10 mM 20 mM 30 mM 40 mM Increasing [S] 19 20 Michaelis-Menten kinetics Relationship between (V0) and [S] 40 mM 30 mM determine initial velocity (V0) for Product [P] each reaction 20 mM 10 mM 20 10  28/03/2023 Michaelis-Menten kinetics Relationship between (V0) and [S] Vmax Reaction Rate (Vo) Substrate Concentration [S] 21 Michaelis-Menten kinetics Relationship between (V0) and [S] Plot of substrate concentration [S] versus initial reaction velocity (V0) Observations Rate of catalysis rises linearly as [S] increases BUT begins to level off and approach a maximum at higher [S] 22 11  28/03/2023 Michaelis-Menten kinetics 23 Almost all enzyme molecules occupied Competition for enzyme molecules Lots of enzyme molecules available 23 Michaelis-Menten Constant (KM) At ½ Vmax [S] = KM 24 12  28/03/2023 25 Michaelis-Menten constant (KM) KM describes the catalytic power of an enzyme KM is the concentration of substrate at which half of the active sites are filled (half Vmax) KM is a measure of the strength of the ES complex (how well S binds to E) High KM indicates high dissociation of ES complex (weak binding) Low KM indicates low dissociation of ES complex (strong binding) 25 Enzymes and cofactors Simple Enzymes Enzymes Apoenzymes Holoenzymes Prosthetic group (conjugated enzymes) Cofactor Coenzymes Cofactor Coenzymes - Loosely-bound organic a non-protein small molecule non-protein cofactors. Eg Vitamins, that binds the enzyme and biotin, Coenzyme A, FAD assist it during catalysis. Prosthetic groups - Tightly-bound Can be organic or inorganic cofactors. Eg Zn2+ , Mg2+ 26 13  28/03/2023 Enzymes and cofactors + Apoenzyme Cofactor Holoenzyme Protein portion Non-protein portion Whole enzyme (Inactive) (Inactive) (Active) 27 Many Enzymes Need Co-factors Example: Succinate dehydrogenase FADH2 Iron/sulphur centres for e-transfer Heme 28 14  28/03/2023 Allosteric Enzymes Have Multiple Binding Sites Effector molecule binds Substrate Inhibitor binds activator Binding of substrate to one active site alters properties of the other active site→ cooperative binding Allosteric enzymes often involved in regulation of biochemical pathways 29 Allosteric Regulation Inhibition Activation 30 15  28/03/2023 31 Allosteric enzymes Do NOT obey Michaelis – Menten kinetics Have multiple subunits and multiple active sites - sigmoidal plot 31 Enzymes a classified based on the type of reaction they catalyse Enzyme names end in "ase“ Oxidoreductases Transferases Isomerases Hydrolases Lyases Ligases 32 32 16  28/03/2023 Oxidoreductases Type of reaction Example Oxidation-reduction Alcohol dehydrogenase (oxidation with NAD+) 33 Transferases Type of reaction Example Transfer of functional groups from one Glycerokinase (phosphorylation) molecule to another 34 34 17  28/03/2023 Isomerases Type of reaction Example Movement of a functional group Maleate isomerase within a molecule (cis-trans isomerization) 35 35 Hydrolases Type of reaction Example Hydrolytic cleavage of one Carboxypeptidase A molecule to two (peptide bon cleavage) 36 36 18  28/03/2023 Lyases Type of reaction Example Addition or removal of groups to break various Pyruvate decarboxylase chemical bonds by means other than hydrolysis 37 37 Ligases Type of reaction Example Joining of two molecules together Pyruvate carboxylase (carboxylation) 38 38 19