SCBC203 Enzymes Jirundon Yuvaniyama, Ph.D. Department of Biochemistry Faculty of Science Mahidol University Gibbs Free Energy of Reaction Free Energy A B + H 2 O A OH + B H Activation Energy Amount of activation energy reduced by enzyme Without Enzyme With Enzyme Enzyme catalyzes a reaction by reducing the activation energy of reaction, and thus increasing the rate of reaction. It does not change the reaction equilibrium and is not consumed during the course of reaction. Adapted from http://chemistry.tutorvista.com/biochemistry/enzyme-kinetics.html Parameters affecting Enzyme Catalysis Substrate concentration ph of the reaction Temperature Amount of enzyme Ionic strength of solution Enzyme Commission umber umerical classification scheme for enzymes, based on the chemical reactions they catalyze: EC 1: Oxidoreductases catalyze redox reactions EC 2: Transferases transfer a functional group EC 3: Hydrolases hydrolyze a covalent bond EC 4: Lyases - addition or removal of groups EC 5: Isomerases - intramolecule rearrangement EC 6: Ligases - join two molecules together with ATP hydrolysis http://en.wikipedia.org/wiki/enzyme_commission_number
Enzyme Commission umber EC 3: Hydrolases hydrolyze a covalent bond EC 3.1: ester bonds (esterases: nucleases, phosphodiesterases, lipase, phosphatase) EC 3.2: sugars (glycosylases/da glycosylases, glycoside hydrolase) EC 3.3: ether bonds EC 3.4: peptide bonds (proteases/peptidases) EC 3.5: carbon-nitrogen bonds, other than peptide bonds EC 3.6: acid anhydrides (acid anhydride hydrolases, including helicases and GTPase) EC 3.7: carbon-carbon bonds EC 3.8: halide bonds EC 3.9: phosphorus-nitrogen bonds EC 3.10: sulfur-nitrogen bonds EC 3.11: carbon-phosphorus bonds EC 3.12: sulfur-sulfur bonds EC 3.13: carbon-sulfur bonds Enzymes with onprotein Components Cofactors May dissociate from enzyme May be further classified as: Metal ions, e.g. Ca 2+, Mg 2+ Coenzymes: small organic compounds some vitamins Prosthestic group Tightly bound to enzyme, e.g. heme in hemoglobin Apoenzyme vs Holoenzyme Apoenzyme: enzyme lacking cofactor Holoenzyme: complete enzyme Enzyme omenclature: http://www.chem.qmul.ac.uk/iubmb/enzyme/ Michaelis Menten Kinetics Michaelis Menten Kinetics Enzyme exists in 2 forms: Free E ES complex One-step reversible binding of E and S to form ES One-step catalysis to release free E and product P [S] V max : maximal rate = k cat x [E] 0 K m : Michaelis Menten equilibrium constant Stability of ES complex = [S] that results in reaction rate of half V max k cat : catalytic rate k cat /K m : catalytic efficiency Figure taken from http://en.wikipedia.org/wiki/michaelis Menten_kinetics Figure taken from https://commons.wikimedia.org/wiki/file:michaelis-menten_saturation_curve_of_an_enzyme_reaction_large.svg
Lineweaver Burk Plot Double reciprocal plot of the Michaelis Menten equation. Useful for graphical presentation and manual linear fitting. However, non-linear regression of the direct plot offers the most accurate estimation of K m and V max. Figure taken from http://en.wikipedia.org/wiki/lineweaver Burk_plot Enzyme Inhibition Binding of inhibitor prevents enzyme s function Irreversible inhibition Inhibitor mostly form covalent adduct with enzyme Reversible inhibition o covalent linkage between enzyme and inhibitor May be divided as Competitive inhibition Uncompetitive inhibition Mixed inhibition oncompetitive inhibition http://en.wikipedia.org/wiki/enzyme_inhibitor Irreversible Inhibition Competitive Inhibition PMSF (phenylmethanesulfonylfluoride) DFP (diisopropylfluorophosphate ) DFP and PMSF are irreversible inhibitors of serine proteases. S and I cannot bind to E at the same time. Can be overcome by sufficiently high [S] out-competing I. V max remains constant, while apparent K m increases (it takes higher [S] to reach half the V max point). Figures taken from http://en.wikipedia.org/wiki/enzyme_inhibitor http://en.wikipedia.org/wiki/pmsf Figures taken from http://en.wikipedia.org/wiki/competitive_inhibition
Competitive Inhibition Uncompetitive Inhibition S and I cannot bind to E at the same time. Can be overcome by sufficiently high [S] out-competing I. V max remains constant, while apparent K m increases (x-intercepts (= 1/K m, app ) get closer to the origin upon increasing [I]). http://en.wikipedia.org/wiki/competitive_inhibition Uncompetitive inhibition occurs when I binds to the ES complex only and not the free form of E. Both K m and k cat (V max ) are affected, resulting in parallel lines in the Lineweaver Burk plot. http://en.wikipedia.org/wiki/uncompetitive_inhibitor Mixed Inhibition on-competitive Inhibition A mixed inhibitor can bind and inhibit the enzyme in both free E and ES complex forms, and may be seen as a conceptual "mixture" of competitive inhibition and uncompetitive inhibition. http://en.wikipedia.org/wiki/mixed_inhibition In the special case of mixed inhibition where α = α, noncompetitive inhibition occurs, in which case V max, app is reduced but K m is unaffected. http://en.wikipedia.org/wiki/on-competitive_inhibition
Pepsin is produced as a zymogen Reaction Specificity Trypsin Chymotrypsin Elastase Propeptide (44 AA) in pepsinogen (proenzyme) suppresses pepsin activity by forcing its active site in an inactive geometry. Lys, Arg Phe, Leu, Ile Gly, Ala, Val Figures taken from http://pdb101.rcsb.org/motm/46 http://en.wikipedia.org/wiki/serine_protease
Catalytic triad Structural Flexibility Structural Flexibility DHF THF ADPH ADPH H 2 O H H H H transfer H H 2 O O O O O As 3D structures of proteins are stabilized by weak interactions, they are flexible and dynamically changing conformation. Some parts of the structure may be more flexible than others and may provide functions. Flexibility of DHFR structure allows for binding of DHF and ADPH as well as its catalysis. O
Isozymes Enzymes that differ in amino-acid sequence but catalyze the same chemical reaction As these are different molecules, they usually have different kinetic parameters (K m and k cat ) or different regulatory properties Examples are lactate dehydrogenase (LDH) isozymes Two forms of subunits: H-form and M-form Different combinations of the two forms are found in various tissues http://en.wikipedia.org/wiki/isozyme Lactate Dehydrogenase Isozymes http://en.wikipedia.org/wiki/isozyme Type Composition Location LDH1 HHHH Heart and Erythrocyte LDH2 HHHM Reticuloendothelial system LDH3 HHMM Brain, kidneys, and lungs LDH4 HMMM Kidneys, placenta, and pancreas LDH5 MMMM Skeletal muscle and liver Allosteric Enzymes An allosteric enzyme possesses allosteric site(s) located further away from the active site, but can affect the catalysis upon binding of its regulatory ligand. on-protein Enzymes RA: Some ribonucleic acids can catalyze the cleavage of phosphodiester bond ribozyme Antibody with catalytic activity abzyme (= antibody + enzyme) or catmab (catalytic monoclonal antibody) Also found in patients with autoimmune diseases such as systemic lupus erythematosus, where they can bind to and hydrolyze DA http://en.wikipedia.org/wiki/allosteric_enzyme http://en.wikipedia.org/wiki/ribozyme http://en.wikipedia.org/wiki/abzyme
Summary Enzyme catalyzes a reaction by reducing the activation energy of reaction Enzyme may contain non-protein components Michaelis Menten kinetics and some enzyme mechanisms Enzyme inhibition Irreversible inhibition Reversible inhibition: competitive, uncompetitive, mixed and noncompetitive inhibitions Isozymes, ribozymes, and abzymes The End