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2014-10-06 ENZYMES as biological catalysts in chemical reactions Outline • Composition, structure and properties of enzyme • How Enzymes work • Enzyme activity • Factors affecting enzyme activity • Regulation of enzyme activities • Enzymes in clinical diagnosis •Coenzymes and cofactors 1 2014-10-06 Three criteria must be met for a chemical reaction to occur: • The reactants, called substrates must collide • The molecular collision must occur with the correct orientation • The reactant must have sufficient energy. This energy is called the activation energy Enzymes are Biological Catalysts Enzymes are proteins that: • Increase the rate of reaction by lowering the energy of activation. • Catalyze nearly all the chemical reactions taking place in the cells of the body. • Have unique three-dimensional shapes that fit the shapes of reactants. 2 2014-10-06 Enzyme Catalyzed Reaction • The proper fit of a substrate (S) in an active site forms an enzyme-substrate (ES) complex. E+S ES • Within the ES complex, the reaction occurs to convert substrate to product (P). ES E+P • The products, which are no longer attracted to the active site, are released. • Overall, substrate is convert to product. E+S ES E+P Enzyme-Substrate Complex • Substrates bind to a depression on the surface of enzymes known as the active site, to form an enzyme-substrate complex. • The active site undergoes a slight conformation change to better accommodate the substrate (induced fit). 3 2014-10-06 Active Site The active site: • Is a region within an enzyme that fits the shape of molecules called substrates. • Contains amino acid R groups that align and bind the substrate. • Releases products when the reaction is complete. Some key features of the active site • The active site takes up a relatively small part of the total volume of an enzyme • The active site is a three-dimensial entity • Substrates are bound to enzymes by multiple weak attraction • Active sites are clefts or crevices • The specificity of binding depends on the precisely defined arrangement of atoms in an active site 4 2014-10-06 The substrate binding site • The substrate binding site is a particular arrangement of chemical groups on the enzyme surface that is specially formulated to bind a specific substrate • The substrate binding site may have been integrated within it the active site or in some cases the active site may not be within the substrate binding site Binding uses multiple weak interactions 1. 2. 3. 4. Hydrogen bonds Salt links van der Waals interactions Hydrophobic effect 5 2014-10-06 The allosteric site • The allosteric site is not at the active site or substrate binding site, but is somewhere else on the molecule • The allosteric site is the site where small molecules bind and affect a change in the active site or the substrate binding site • The binding of this specific molecule causes a change in the conformation of enzyme what cause the active site to become either more or less active • It may cause the binding site to have a greater affinity for substrate or have less affinity for substrate Enzyme Specificity Enzymes may recognize and catalyze: • A single substrate. • A group of similar substrates. • A particular type of bond. 6 2014-10-06 •Trypsin, chymotrypsin, and elastase all carry out the same reaction — the cleavage of a peptide chain, but they display very different specificities. •Trypsin cleaves peptides on the carbonyl side of the basic amino acids, arginine or lysine. •Chymotrypsin prefers to cleave on the carbonyl side of aromatic residues, such as phenylalanine and tyrosine. • Elastase is not as specific as the other two; it mainly cleaves peptides on the carbonyl side of small, neutral residues. Classification of Enzymes • Enzymes are classified according to the reaction they catalyze. Class Reactions catalyzed 1. Oxidoreductases Oxidation-reduction 2. Transferases Transfer groups of atoms 3. Hydrolases Hydrolysis 4. Lyases Add atoms/remove atoms to/from a double bond 5. Isomerases Rearrange atoms 6. Ligases Use ATP to combine molecules 7 2014-10-06 The following classification names apply: • • • • • Oxidoreductases These are enzymes that catalyze oxidations or reductions. Enzymes such as dehydrogenases, oxidases, and peroxidases. • Transferases These enzymes catalyze the transfer of a group from one molecule to another. Examples such as phosphatases, transaminases, and transmethylases. Classification of Enzymes: Oxidoreductases and Transferases 8 2014-10-06 • • • • • Hydrolases These enzymes catalyze hydrolysis reactions. Examples are the digestive enzymes such as sucrase, amylase, maltase, and lactase. • Lyases These enzymes catalyze the removal of groups in non-aqueous media. An example would be the decarboxylases. Classification: Hydrolases and Lyases 9 2014-10-06 • Isomerases • Enzymes that catalyze the isomerization of molecules. • Examples are racemases, and cis-trans isomerases. • Ligases • These are also called synthetases which are enzymes that catalyze condensation reactions where smaller molecules are connected with the resulting removal of a water molecule. • This is accompanied with the formation of a high energy phosphate link that stores energy. • An example would be the amino acid RNA ligases. Classification: Isomerases and Ligases 10 2014-10-06 Two Models for Enzyme-Substrate Interaction Lock-and-Key Model In the lock-and-key model of enzyme action: • The active site has a rigid shape. • Only substrates with the matching shape can fit. • The substrate is a key that fits the lock of the active site. Induced-fit Model In the induced-fit model of enzyme action: • The active site is flexible, not rigid. • The shapes of the enzyme, active site, and substrate adjust to maximum the fit, which improves catalysis. • There is a greater range of substrate specificity. 11 2014-10-06 Induced Conformational Change in Hexokinase The Michaelis-Menten equation is the basic equation of enzyme kinetics • For many enzymes, the rate of catalysis, V varies with substrate concentration, [S], in a following manner Vmax [S ] V= (K m + [S ]) Km describes the substrate concentration at which the reaction velocity is halfmaximal Vmax occurs at high substrate concentrations when the enzyme is sturated, that is, when it is entirely in the ES form 12 2014-10-06 It is convenient to transform the M-M equation into one that gives a straight line plot Vmax [S ] V= (K m + [S ]) • Simplifying the above equation gives the Lineweaver-Burk relationship • Taking the reciprocal of both sides of the equation yields Plotting the reciprocals of the same data points yields a "double-reciprocal" or Lineweaver-Burk plot. This provides a more precise way to determine Vmax and Km. 13 2014-10-06 Plots of 1/v versus 1/[S] yield straight lines having a slope of Km/Vmax and an intercept on the ordinate at 1/Vmax Factors affecting enzyme activity • • • • • • Concentration of substrate Concentration of enzyme Temperature pH Activators Inhibitors 14 2014-10-06 Substrate Concentration • The rate of reaction increases as substrate concentration increases (at constant enzyme concentration). • Maximum activity occurs when the enzyme is saturated. pH and Enzyme Action Enzymes: • Are most active at optimum pH. • Contain R groups of amino acids with proper charges at optimum pH. • Lose activity in low or high pH as tertiary structure is disrupted. 15 2014-10-06 Optimum pH Values • Most enzymes of the body have an optimum pH of about 7.4. • In certain organs, enzymes operate at lower and higher optimum pH values. Temperature and Enzyme Action Enzymes: • Are most active at an optimum temperature (usually 37°C in humans). • Show little activity at low temperatures. • Lose activity at high temperatures as denaturation occurs. 16 2014-10-06 Temperature and Enzyme Action • As the temperature rises, reacting molecules have more and more kinetic energy. • This increases the chances of a successful collision and so the rate increases. • Above this temperature the enzyme structure begins to break down (denature) since at higher temperatures intra- and intermolecular bonds are broken as the enzyme molecules gain even more kinetic energy. Enzyme Concentration • The rate of reaction increases as enzyme concentration increases (at constant substrate concentration). • At higher enzyme concentrations, more substrate binds with enzyme. 17 2014-10-06 TERMINOLOGY • Enzyme activity is usually expressed in units of µmol of substrate converted to product per minute under specific assay conditions • One standard unit of enzyme activity (U) is an amount of activity that catalyzes the transformation of 1 µmol/min • The specific activity is defined as the number of enzyme units per mg of protein (U/mg of protein) • The catalytic constant Kcat or turnover number is equal to the units of enzyme activity per mol of enzyme • Katal (kat) denotes the conversion of 1 mol substrate per second • The maximum velocity Vmax is the velocity obtained under condition of substrate saturation of the enzyme under a given set of condition of pH, temperature, Enzyme Inhibition • Inhibitors are molecules that reduce the rate of enzymatic reactions • The are usually specific and they work at low concentrations • They block the enzyme but they do not usually destroy it • Many drugs and poisons are inhibitors of enzymes in the nervous system 18 2014-10-06 Types of inhibitors • Enzyme Inhibitors can be divided into two major types: Reversible inhibitors and irreversible inhibitors Reversible Versus Irreversible Inhibition • Reversible inhibitors interact with the enzyme through noncovalent association/dissociation reactions. • In contrast, irreversible inhibitors usually cause stable, covalent alterations in the enzyme. • That is, the consequence of irreversible inhibition is a decrease in the concentration of active enzyme. 19 2014-10-06 The effect of enzyme inhibition 1. Competitive: • Inhibitor competes with the substrate molecules for the E+I active site of enzyme Reversible • The inhibitor’s action is proportional to its concentration reaction • Inhibitor resembles the substrate’s structure closely Has its effect reversed by increasing substrate concentration. EI Enzyme inhibitor complex Double Reciprocal Plots Facilitate evaluation of inhibitors Figure represents a typcal case of competitive inhibition shown graphically in the form of Lineweaver-Burk plot Competitive inhibitor increases Km but not changes Vmax 20 2014-10-06 Malonate and Succinate Dehydrogenase Succinate Succinate dehydrogenase Fumarate + 2H++ 2e- Malonate: • Is a competitive inhibitor of succinate dehydrogenase. • Has a structure that is similar to succinate. • Inhibition is reversed by adding succinate. Ethanol as inhibitor in methanol and ethylene glycol poisonings Methanol is relatively non-toxic; however, it is metabolized to highly toxic compounds that are responsible for the acidosis and blindness characteristic of methanol poisoning. Ethanol is a competing substrate and so it blocks the oxidation of methanol to aldehyde products 21 2014-10-06 Noncompetitive Inhibition A noncompetitive inhibitor: • Has a structure different than the substrate. • Distorts the shape of the enzyme, which alters the shape of the active site. • Prevents the binding of the substrate. • Cannot have its effect reversed by adding more substrate. Noncompetitive Inhibition Lineweaver-Burk plot for reversible noncompetitive inhibition Noncompetitive inhibitor decreases Vmax but not changes Km 22 2014-10-06 Uncompetitive inhibition. In uncompetitive inhibition inhibitor binds only to enzyme-substrate complex. Uncompetitive inhibition decreases the maximum velocity (Vmax) as well as the KM Irreversible Inhibition • In irreversible inhibition, a substance destroys enzyme activity by bonding with side-chain groups in(R) the active site . 23 2014-10-06 Regulation of enzyme activity Regulation of enzyme activity is achieved in a variety of ways: • ranging from controls over the amount of enzyme protein produced by the cell • to more rapid, reversible interactions of the enzyme with metabolic inhibitors and activators. Zymogens • Zymogen: Inactive enzyme precursor, cleavage of one or more covalent bonds transforms it into active enzyme • Chymotrypsinogen – synthesized and stored in the pancreas – a single polypeptide chain of 245 amino acid residues cross linked by 5 disulfite bonds – when secreted into the small intestine, the digestive enzyme trypsin cleaves a 15 unit polypeptide from the N-terminal end to give α chymotrypsin (active form) 24 2014-10-06 Digestive Enzymes Digestive enzymes are: • Produced as zymogens in one organ and transported to another such as the pancreas when needed. • Activated by removing small peptide sections. Allosteric Enzymes • Allosteric: Greek allo + steric, other shape • Allosteric enzyme: an oligomer whose biological activity is affected by other substances binding to it – these substances change the enzyme’s activity by altering the conformation(s) of its 4°structure • Allosteric effector: a substance that modifies the behavior of an allosteric enzyme; may be an – allosteric inhibitor – allosteric activator 25 2014-10-06 Allosteric Enzymes • An allosteric enzyme is an enzyme in a reaction sequence that binds a regulator substance. • A positive regulator enhances the binding of substrate and accelerates the rate of reaction. • A negative regulator prevents the binding of the substrate to the active site and slows down the rate of reaction. A change in shape • When the inhibitor is present it fits into its site and there is a conformational change in the enzyme molecule • The enzyme’s molecular shape changes • The active site of the substrate changes • The substrate cannot bind with the active site 26 2014-10-06 Switching off • These enzymes have two receptor sites • One site fits the substrate like other enzymes • The other site fits an inhibitor molecule Substrate cannot fit into the active site Inhibitor molecule Inhibitor fits into allosteric site Phosphorylation /dephosphorylation •most common covalent modification • involve protein kinases/phosphatase •PDK (pyruvate dehydrogenase) inactivated by phosphorylation •Amino acids with –OH groups are targets for phosphorylation 27 2014-10-06 Feedback Control In feedback control: • A product acts as a negative regulator. • An end product binds with the first enzyme (E1) in a sequence, when sufficient product is present. Diagnostic Enzymes • The levels of diagnostic enzymes determine the amount of damage in tissues. 28 2014-10-06 Isoenzymes • Isoenzymes are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. • These enzymes usually display different kinetic parameters (e.g. different KM values), or different regulatory properties • In clinical medicine, isoenzyme has a more restricted meaning, namely, the physically distinct and separable forms of a given enzyme present in different cell types or subcellular compartments of a human being. Lactate dehydrogenase (LDH) • Lactate dehydrogenase isoenzymes differ at the level of quaternary structure • The oligometric LDH molecule consists of 4 protomers of 2 types, H and M. • Only the tetrameric molecule possesses catalytic activity. 29 2014-10-06 Isoenzymes • Isoenzymes catalyze the same reaction in different tissues in the body. • Lactate dehydrogenase, which converts lactate to pyruvate, (LDH) consists of five isoenzymes. Enzyme Cofactors • • • A simple enzyme is an active enzyme that consists only of protein. Many enzymes are active only when they combine with cofactors such as metal ions or small molecules. A coenzyme is a cofactor that is a small organic molecule such as a vitamin. 60 30 2014-10-06 Metal ion catalysts One-third of all known enzymes needs metal ions to work!! 1. Metalloenzymes: contain tightly bound metal ions: I.e. Fe++, Fe+++, Cu++, Zn++, Mn++, or Co++. 2. Metal-activated enzymes- loosely bind ions Na+, K+, Mg++, or Ca++. They participate in one of three ways: They bind substrates to orient then for catalysis Through redox reactions gain or loss of electrons. Electrostatic stabilization or negative charge shielding Vitamin Coenzymes Vitamin Thiamine (B1) Riboflavin (B2) Coenzyme Made Thiamine Pyrophosphate FAD, FMN folic acid tetrahydrofolic acid biotin biocytin Function Decarboxlation Electron Transfer amino acid metabolism CO2 fixation Pantothenic Acid Coenzyme A acyl group carrier Ascorbic Acid (C) Vitamin C Collagen synthesis Healing 31