Lecture 2 - Enzymes.pdf

 Enzymes 1) What is an Enzyme? 2) The molecular nature of an enzyme? 3) How does it work?  What is an Enzyme? Enzymes are a biological catalyst. – Catalytic power: They speed up a chemical reaction – carbonic anhydrase: In fact, carbonic anhy...

 Enzymes 1) What is an Enzyme? 2) The molecular nature of an enzyme? 3) How does it work?  What is an Enzyme? Enzymes are a biological catalyst. – Catalytic power: They speed up a chemical reaction – carbonic anhydrase: In fact, carbonic anhydrase is one of the fastest enzymes known. Each enzyme molecule can hydrate 106 molecules of CO2 per second. This catalyzed reaction is 107 times as fast as the uncatalyzed one.  What is an Enzyme? – Specificity: reactions they catalyze and substrates (reactants) – Specific or similar reactions- peptide bonds (papain, Thrombin (arg- gly)) – The specificity of an enzyme is due to the precise interaction of the substrate with the enzyme. This precision is a result of the intricate three-dimensional structure of the enzyme protein. – Proteins or Some RNA molecules have enzymatic activity (ribozyme)  What is an Enzyme? Simple enzymes are composed completely of protein. Conjugated enzymes are composed of an apoenzyme (protein portion) + cofactors Cofactor: cofactors are able to execute chemical reactions that cannot be performed by the standard set of twenty amino acids that make enzyme. – inorganic metal ion (e.g. Fe, Mg Zn). – Coenzyme = organic compounds (e.g. vitamins, NAD, NADP, FAD) Aponenzyme + cofactor = holoenzyme (complete catalytically active enzyme) Tightly bound coenzymes are called prosthetic groups Basic currency of cell to store energy is ATP.  Essential Factors Metal ion Substrate Co-enzyme Co-factor Temperature pH Metal ions as Common Cofactors Common Coenzymes Enzyme Classification or ring  An enzyme is called oxidoreductase if it catalyzes: 1.Group transfer reactions 2.Hydrolysis reactions 3.Transfer of electrons 4.Addition of groups to double bonds pH Activity Profiles of Two Enzymes Substrate Binding to Enzyme at the Active Site  Common features of active sites The active site takes up relatively little of the enzyme. The active site is a 3D surface. Substrates are bound to enzymes by multiple weak attractions. Active sites are clefts or crevices. Specificity depends on the precise arrangement of atoms in the active site. – Lock-and-key – Induced fit  Lock-and-key model of enzyme–substrate binding. In this model, the active site of the unbound enzyme is complementary in shape to the substrate. Complementary shapes of a substrate and active site (Dihydrofolate reductase, NADP+, and tetrahydrofolate)  Induced-fit model of enzyme–substrate binding. In this model, the enzyme changes shape on substrate binding. The active site forms a shape complementary to the substrate only after the substrate has been bound.  Active site crevice Strong interactions ES complex: Covalent bonds Weak interactions ES complex: hydrogen bonds, Van der Waals forces, Electrostatic interaction. Debler E W et al. PNAS 2009;106:18539-18544 The architecture of the binding pocket reveals the structural basis for the observed substrate specificity. Enzymes can transform energy from one form into another photosynthesis cellular respiration enzymes can then use the chemical-bond energy of ATP in diverse ways. -myosin- ATP to mechanical energy -Pumps-ATP to transport molecules Enzymes speed up the rate of chemical reactions, but the properties of the reaction—whether it can take place at all and the degree to which the enzyme accelerates the reaction—depend on energy differences between reactants and products.  Gibbs free energy: Thermodynamic function to understand enzymes Gibbs free energy (G), is a thermodynamic property that is a measure of useful energy, or the energy that is capable of doing work. Enzymes reactions depends on two thermodynamic properties – Free energy difference ∆G between products and reactants-spontaneous reactions – the energy required to initiate the conversion of reactants into products, it determines the rate of the reaction- affect by presence of enzymes  Gibbs free energy change A reaction can take place spontaneously only if ∆G is negative_exergonic. A system is at equilibrium and no net change can take place if ∆G is zero. A reaction cannot take place spontaneously if ∆G is positive_endothermic The ∆G of a reaction depends only on the free energy of the products (the final state) minus the free energy of the reactants (the initial state). ∆G provides provides information about spontaneity but not the rate of reaction Rate of reaction depends on free energy of activation. The difference in free energy between the transition state and the substrate is called the Gibbs free energy of activation Reaction Coordinate Diagram for a Chemical Reaction ΔG < 0; favors reaction (Spontaneous). Negative ΔG favors the reaction, these reactions are called exergonic ΔG = 0; Neither the forward nor the reverse reaction prevails (Equilibrium) ΔG > 0; disfavors reaction (Nonspontaneous). These reactions are termed endergonic. -The ΔG provides information about feasibility of reaction but not about the rate of a reaction Reaction Kinetics A B C  Role of Binding Energy in Catalysis -Enzymes lower the activation energy -Enzymes lower the amount of energy needed to complete the reaction by lowering the activation energies of the transition states. -Free energy is released by the formation of a large number of weak interactions between a complementary enzyme and its substrate. The free energy released on binding is called the binding energy. Enzymes affect reaction rates, not Equilibria A+B AB A+B + E AB + E E=Enzyme Enzymes alter only the reaction rate and not the reaction equilibrium Note that the amount of product formed is the same whether or not the enzyme is present but, in the current example, the amount of product formed in seconds when the enzyme is present might take hours  Determining the relation between initial velocity and substrate concentration The study of the rates of chemical reactions is called kinetics, and the study of the rates of enzyme-catalyzed reactions is called enzyme kinetics. A) The amount of product formed at different substrate concentrations is plotted as a function of time. The initial velocity ( V 0 ) for each substrate concentration is determined from the slope of the curve at the beginning of a reaction, when the reverse reaction is insignificant. (B) The values for initial velocity determined in part A are then plotted, with error bars, against substrate concentration. (C) The data points are connected to clearly reveal the relationship of initial velocity to substrate concentration. V0, or the initial rate of catalysis, as the number of moles of product formed per second when the reaction is just beginning—that is, when t ≈ 0  Substrate Concentration on Reaction Velocity -The maximal rate, V max , is attained when the catalytic sites on the enzyme are saturated with substrate—that is, when [ ES ] = [ E ]T (total enzyme conc.) -A high KM , means Enzyme achieves a high rate of catalysis only at very high concentrations of substrate means low affinity for its substrate.  Interpreting Km Km (Michaelis constant) represents the concentration of substrate at which the reaction proceeds at its ½ maximal velocity. Km is used to compare the affinity of an enzyme for various substrates. The lower the concentration of substrate needed to reach the ½ maximal velocity the more readily it binds the enzyme. Km can vary greatly from enzyme to enzyme, and even for different substrates of the same enzyme.  Enzyme Inhibition Inhibition can be reversible or non-reversible (i.e., permanent) The reversible inhibition of enzymes in biological systems provides a rapid regulation of the enzymes activity and therefore the balance of reactants and products in the cell Non-reversible inhibition involves degradation or covalent modification of the enzyme preventing its function (e.g., modification by toxins)  Three Types of Reversible Inhibition Penicillin irreversibly inactivates a key enzyme in bacterial cell-wall synthesis. Penicillin inhibits the cross-linking transpeptidase by the Trojan horse stratagem.  Distinction between reversible inhibitors (A) Enzyme–substrate complex; (B) a competitive inhibitor binds at the active site and thus prevents the substrate from binding; (C) an uncompetitive inhibitor binds only to the enzyme–substrate complex; (D) a noncompetitive inhibitor does not prevent the substrate from binding. Kinetics of Inhibitors  Regulation of Allosteric enzymes 1. Allosteric Enzymes Undergo Conformational Changes in Response to Regulator Binding (Activation and repression) 2. Feedback regulation  Kinetics of Allosteric Enzyme-Sigmoidal An important group of enzymes that do not obey Michaelis–Menten kinetics are the allosteric enzymes. These enzymes consist of multiple subunits and multiple active sites. Two Views of the Regulatory Enzyme Aspartate-Transcarbamylase  Feedback Inhibition What is the feedback inhibitor for enzyme threonine deaminase? 1. Threonine 2. isoleucine Feedback Regulation and Feedback Inhibition Enzyme-Modification Reactions Regulation of Glycogen Phosphorylase Activity by Covalent Modification