Enzymes Lecture Notes

6/21/2024 Enzymes Lecture Outline Enzymes and Their Substrates Structure of enzymes Enzyme Specificity Thermodynamics of Chemical Reactions Enzymes and Catalysis Naming Enzymes Enzymes Classification Factors That Affect Enzyme Activity...

6/21/2024 Enzymes Lecture Outline Enzymes and Their Substrates Structure of enzymes Enzyme Specificity Thermodynamics of Chemical Reactions Enzymes and Catalysis Naming Enzymes Enzymes Classification Factors That Affect Enzyme Activity 2 1 Lecturer. KHALED HREEBA  6/21/2024 Introduction The union of nitrogen and hydrogen to form ammonia in a chemical process requires a high temperature of 450°C and a pressure of 200 atm, while ammonia is also formed by the union of nitrogen and hydrogen at a temperature of 25°C and under normal atmospheric pressure through bacterial cells in the roots of legumes, a process of nitrogen fixation aerial. N2 + 3H2 2NH3 3 When sucrose is decomposed in the laboratory into its building units (glucose + fructose), it is boiled with water in an acidic medium as a source of hydrogen, while sucrose is decomposed in the small intestine of humans at body temperature and under moderate conditions. In the previous reactions, it is noted that when performed in the laboratory, high temperature and pressure are required, in addition to the use of chemicals to increase the speed of these reactions to reach the required equilibrium state. The chemicals used are called chemical cofactors. These previous reactions also occur in living cells under moderate conditions of temperature and hydrogen ion concentration and at a tremendous speed with the help of biochemical factors in cells called enzymes. 4 2 Lecturer. KHALED HREEBA  6/21/2024 Therefore, enzymes are vital cofactors present in cells that lower the activation energy, which allows the completion of reactions that usually take place at very high temperatures, according to the vital conditions at a temperature that does not exceed the temperature of the living body, to return after the completion of the reaction to its original position, which enables it to participation in a new reaction. This allows small amounts of them to participate for a long period of time in the reactions. It is known that within the tissues and cells of the living organism, thousands of diverse and different biological reactions occur, such as oxidation, reduction, building, demolition, transport and decomposition reactions. The organism depends mainly on it in its growth, development, movement and reproduction, and the course of these reactions takes place under the influence and with the help of enzymes. 5 Definition of enzymes: They are biological cofactors that accelerate the rates of biochemical reactions. Enzymes are protein substances with high molecular weights that are synthesized inside the living cell in order to help increase the speed of the vital reactions that occur inside these cells and upon which living organisms depend for their various activities. The amino acids are arranged in the peptide chains that make up the enzymes according to a specific sequence to each enzyme, which ultimately leads to a specific three-dimensional space structure that enables the enzyme to accelerate the occurrence of its own reaction. The enzyme is a Latin word meaning "in yeast" and was first discovered in the process of fermenting glucose into alcohol by yeast. 6 3 Lecturer. KHALED HREEBA  6/21/2024 In the body of the living organism there are large numbers of enzymes, each of which has a special substrate that fits perfectly with it, and one enzyme molecule can perform its full work a million times per minute, and the reaction occurs in the presence of the enzyme at a speed that exceeds the speed of its occurrence without the enzyme by thousands and even millions of times. Enzymes are similar in action to other chemical cofactors. It participates in the reaction without changing its result, that is, at the end of the reaction, it returns to its original position before the start of the reaction, but it is distinguished from other chemical cofactors in its high efficiency. 7 It is also distinguished from other catalysts by its high degree of specialization with respect to the reactant and the type of reaction. Each enzyme can be concerned with one reactant substance called the substrate, and the enzyme may be concerned with a specific group of substances that are similar in structure. Examples of enzymes differing according to the different substrate are numerous, among which are mentioned the hydration of the glycosidic bond, the ester bond, or the peptide bond in carbohydrate, lipid and protein molecules, respectively. In all these reactions, the bond is broken by adding a molecule of water, where a hydroxyl group -OH is added to one of the two atoms, while a hydrogen atom -H is added to the other atom. Although the reactions are similar in the three cases, the enzymes differ according to the target. 8 4 Lecturer. KHALED HREEBA  6/21/2024 Enzymes play an important role in Metabolism, Diagnosis, and Therapeutics. All biochemical reactions are enzyme catalyzed in the living organism. Level of enzyme in blood are of diagnostic importance e.g. it is a good indicator in disease such as myocardial infarction. Enzyme can be used therapeutically such as digestive enzymes. Properties of enzymes Reaction specific each enzyme works with a specific substrate chemical fit between active site & substrate H bonds & ionic bonds Not consumed in reaction single enzyme molecule can catalyze thousands or more reactions per second enzymes unaffected by the reaction Affected by cellular conditions any condition that affects protein structure temperature, pH, salinity 5 Lecturer. KHALED HREEBA  6/21/2024 Enzymes work in an aqueous environment in our body so that the protein chain folds such that the polar amino acids are on the surface. Consider hexokinase, an enzyme whose job is to transfer a phosphate group from the high energy molecule, adenosine triphosphate, ATP, to D-glucose. In this equation, the enzyme name is written above or below the reaction arrow. The phosphate group is represented by a P in a circle. 11 The Active Site The folded structure for hexokinase is shown here. 12 Chapter 10 6 Lecturer. KHALED HREEBA  6/21/2024 Enzymes and Their Substrates: When in its proper three-dimensional shape, hexokinase has an indentation on one side of the structure. This indentation is known as the active site, and it is lined with amino acid side chains. The active site is the functional part of an enzyme where catalysis occurs. Glucose, the reactant for hexokinase, fits snugly in the active site. In an enzyme reaction, the reactant is called the substrate. Enzymes have specific substrates, a property known as substrate specificity. For example, the active site of hexokinase reacts with D-glucose, but will not react with L-glucose. Enzymes are specific for one enantiomer of the substrate. 13 14 7 Lecturer. KHALED HREEBA  6/21/2024 Enzyme Specificity Enzymes have varying degrees of specificity for substrates Enzymes may recognize and catalyze: - a single substrate - a group of similar substrates - a particular type of bond 8 Lecturer. KHALED HREEBA  6/21/2024 Structure of enzymes Enzymes are a linear chain of amino acids, which give rise to a three-dimensional structure. The sequence of amino acids specifies the structure, which in turn identifies the catalytic activity of the enzyme. Upon heating, the enzyme’s structure denatures, resulting in a loss of enzyme activity, which typically is associated with temperature. Compared to its substrates, enzymes are typically large with varying sizes, ranging from 62 amino acid residues to an average of 2500 residues found in fatty acid synthase. Only a small section of the structure is involved in catalysis and is situated next to the binding sites. The catalytic site and binding site together constitute the enzyme’s active site. APOENZYME and HOLOENZYME The enzyme without its non protein moiety is termed as apoenzyme and it is inactive. Holoenzyme is an active enzyme with its non protein component. 9 Lecturer. KHALED HREEBA  6/21/2024 Some enzymes, like hexokinase, have non-protein helpers. Two categories of helpers are as follows: 1. Cofactors are inorganic substances like magnesium ions. 2. Coenzymes are small organic molecules derived from vitamins. Riboflavin found in the coenzyme flavin adenine dinucleotide (FAD) is a coenzyme. 19 Structure of enzymes Enzymes Complex or holoenzymes (protein part Simple (only protein) and nonprotein part – cofactor) Apoenzyme (protein part) Cofactor Prosthetic groups Coenzyme usually small inorganic - -large organic molecule or atom; molecule usually tightly bound to - -loosely bound to apoenzyme apoenzyme 10 Lecturer. KHALED HREEBA  6/21/2024 Enzyme–Substrate Models A substrate is drawn into the active site by intermolecular attractions like hydrogen bonding. Hydrogen bonding orients the substrate properly within the active site. The initial interaction of the enzyme with the substrate is called the enzyme–substrate complex (ES). This complex forms prior to catalysis. 21 There are two enzyme–substrate models: 1. In the Lock-and-key model, the active site is thought to be a rigid, inflexible shape that is an exact complement to the shape of the substrate. The substrate fits in the active site much like a key fits in a lock. 2. In the induced-fit model, the active site is flexible, has a shape roughly complementary to the shape of its substrate, and undergoes a conformational change, adjusting to the shape of the substrate when the substrate interacts with the enzyme. 22 11 Lecturer. KHALED HREEBA  6/21/2024 Enzymes and Their Substrates, Continued 23 Enzymes and Their Substrates, Continued A good example of an induced-fit model is when hexokinase and glucose form an enzyme–substrate complex as shown. 24 12 Lecturer. KHALED HREEBA  6/21/2024 Enzyme Catalyzed Reactions When a substrate (S) fits properly in an active site, an enzyme- substrate (ES) complex is formed: E + S  ES Within the active site of the ES complex, the reaction occurs to convert substrate to product (P): ES → E + P The products are then released, allowing another substrate molecule to bind the enzyme - this cycle can be repeated millions (or even more) times per minute The overall reaction for the conversion of substrate to product can be written as follows: E + S  ES → E + P Step 1: Enzyme-substrate complex Enzyme and substrate combine to form complex E + S ES Enzyme Substrate Complex + 13 Lecturer. KHALED HREEBA  6/21/2024 Step 2: Enzyme-product complex An enzyme-product complex is formed. ES EP ES transition EP state Step 3: Product The enzyme and product separate EP E + P The product is made Enzyme is ready EP for another substrate. 14 Lecturer. KHALED HREEBA  6/21/2024 Thermodynamics of Chemical Reactions As chemical reactions occur, some bonds are formed and some are broken, and in the process, the amount of energy changes. Some reactions release energy as heat (exothermic reactions), and some absorb energy as heat (endothermic reactions). A collision of reactant molecules must occur for a chemical reaction to occur. 29 In an exothermic reaction, the energy of reactants is higher than the energy of products, so heat is released. In an endothermic reaction, the energy of products is higher than the energy of reactants, so heat is absorbed. Reactions with a low activation energy will proceed at a faster rate than reactions with a high activation energy. Activation energy can be lowered with a catalyst, which will cause the reaction to proceed at a faster rate. 30 15 Lecturer. KHALED HREEBA  6/21/2024 An enzyme-catalyzed reaction increases the rate of a reaction by forming ES before forming a product. 31 How do they work? lower activation energy & speed up reaction active site orients substrates in correct position for reaction enzyme brings substrate closer together active site binds substrate & puts stress on bonds that must be broken, making it easier to separate molecules 16 Lecturer. KHALED HREEBA  6/21/2024 Proximity The reacting molecules are in close proximity to each other, and the closer they are the more likely a reaction will occur. Orientation Amino acid side chains in the active site create interactions that orient the substrates. This lowers the activation energy needed for the reaction to occur. When an enzyme interacts with substrate to form ES, the bonds of the substrate molecule are weakened (strained). Strained bonds in the substrate means that the reaction will proceed more rapidly because the activation energy is lowered by this effect. 33 Enzymes and Catalysis, Continued: Proper orientation is shown as: 34 17 Lecturer. KHALED HREEBA  6/21/2024 Consider the hexokinase reaction that catalyzes glucose to glucose-6-phosphate. Mg2+ (a coenzyme) holds ATP in one area of the active site and glucose interacts with another area. Amino acid side chains in the active site form multiple hydrogen bonds with glucose, which stabilizes ES and lowers the activation energy. A conformational change in the enzyme occurs when glucose enters the active site. ATP is in close proximity to the glucose and is in proper orientation for the reaction. Glucose-6-phosphate is formed along with ADP. The enzyme is less attracted to the products, so the enzyme move and the products are released. 35 This figure shows the formation of glucose-6-phosphate by hexokinase. 36 18 Lecturer. KHALED HREEBA  6/21/2024 Enzymes Lower a Reaction’s Activation Energy Naming Enzymes The name of an enzyme in many cases end in –ase For example, sucrase catalyzes the hydrolysis of sucrose The name describes the function of the enzyme For example, oxidases catalyze oxidation reactions Sometimes common names are used, particularly for the digestion enzymes such as pepsin and trypsin Some names describe both the substrate and the function For example, alcohol dehydrogenase oxides ethanol 19 Lecturer. KHALED HREEBA  6/21/2024 Naming Enzymes: Principle of the international classification Each enzyme has classification number consisting of four digits: Example, EC:(2.7.1.1) D-Hexose-6-Phosphotransferase (HEXOKINASE) EC: (2.7.1.1) these components indicate the following groups of enzymes: 2. IS CLASS (TRANSFERASE) 7. IS SUBCLASS (TRANSFER OF PHOSPHATE) 1. IS SUB-SUB CLASS (ALCOHOL IS PHOSPHATE ACCEPTOR) 1. SPECIFIC NAME ATP,D-HEXOSE-6 PHOSPHOTRANSFERASE (Hexokinase) Enzymes Classification Enzymes Are Classified into six functional Classes (EC number Classification) by the International Union of Biochemists (I.U.B.). on the Basis of the Types of Reactions That They Catalyze EC 1. Oxidoreductases EC 2. Transferases EC 3. Hydrolases EC 4. Lyases EC 5. Isomerases EC 6. Ligases 20 Lecturer. KHALED HREEBA  6/21/2024 Enzymes Classification EC 1.Oxidoreductases The enzyme Oxidoreductase catalyzes the oxidation and reduction reaction where the electrons tend to travel from one form of a molecule to the other. alcohol dehydrogenase, catalyzing the oxidation of ethanol to acetaldehyde. CH3CH2OH + NAD+ alcohol dehydrogenase CH3CHO + NADH + H+ succinate dehydrogenase, catalyzing the oxidation of succinate to fumarate. Enzymes Classification EC 2.Transferases These catalyze transferring of the chemical group from one to another compound. An example is a hexokinase, which transfers an phosphate group from ATP to glucose. 21 Lecturer. KHALED HREEBA  6/21/2024 An example is a transaminase, which transfers an amino group from one molecule to another. Enzymes Classification EC 3.Hydrolases Hydrolases are hydrolytic enzymes, which catalyze the hydrolysis reaction by adding water to cleave the bond and hydrolyze it. For example, the enzyme sucrase hydrolyzes glycosidic bond in sucrose. sucrase 22 Lecturer. KHALED HREEBA  6/21/2024 Enzymes Classification EC 4.Lyases These catalyse the breakage of bonds without catalysis, adds water, carbon dioxide or ammonia across double bonds or eliminate these to create double bonds. An example is an aldolase (an enzyme in glycolysis) catalysis the splitting of fructose-1, 6-bisphosphate to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Enzymes Classification EC 5.Isomerases The Isomerases enzymes catalyze the structural shifts present in a molecule, thus causing the change in the shape of the molecule. 23 Lecturer. KHALED HREEBA  6/21/2024 Enzymes Classification EC 6.Ligases The Ligases enzymes are known to charge the catalysis of a ligation process. Isoenzymes are different forms of an enzyme that catalyze the same reaction in different cells or tissues of the body. have quaternary structures with slight variations in the amino acids in the polypeptide subunits. The different forms of an enzyme allow a medical diagnosis of damage or disease to a particular organ or tissue. Myocardial infarction may be indicated by an increase in the levels of creatine kinase (CK) and lactate dehydrogenase (LDH). 24 Lecturer. KHALED HREEBA  6/21/2024 LACTATE DEHYDROGENASE (LDH) Pyruvate Lactate (anaerobic glycolysis) LDH is elevated in myocardial infarction, blood disorders It is a tetrameric protein and made of two types of subunits namely H = Heart, M = skeletal muscle It exists as 5 different isoenzymes with various combinations of H and M subunits The different isoenzymes of lactate dehydrogenase (LDH) indicate damage to different organs in the body. Isoenzyme Composition Composition Present in Elevated in name LDH1 ( H4) HHHH Heart, RBC myocardial infarction LDH2 (H3M1) HHHM Brain, RBC, Heart myocardial infarction LDH3 (H2M2) HHMM Brain, Lung, Leukemia Kidneys LDH4 (H1M3) HMMM Skeletal muscle, Viral hepatitis Liver LDH5 (M4) MMMM Skeletal muscle, Skeletal muscle Liver and liver diseases 25 Lecturer. KHALED HREEBA  6/21/2024 CREATINE KINASE (CK) Creatine + ATP phosphocreatine + ADP (Phosphocreatine – serves as energy reserve during muscle contraction) Creatine kinase is a dimer made of 2 monomers occurs in the tissues Skeletal muscle contains M subunit, Brain contains B subunits Three different isoenzymes are formed 51 Isoenzyme Composition Present in Elevated in name CK-1 BB Brain CNS diseases )Central nervous system disease( Myocardium/ Acute myocardial CK-2 MB Heart infarction Skeletal muscle, CK-3 MM Myocardium 26 Lecturer. KHALED HREEBA  6/21/2024 Factors That Affect Enzyme Activity If allowed the slices of apples untouched, they will turn brown by a process known as oxidation, caused by an enzyme. If lemon juice is sprinkled on the sliced apple, the vitamin C in the lemon juice will inhibit the formation of this brown color by changing the pH of the environment of the enzyme. Enzyme reactions are affected by reaction conditions such as substrate concentration, pH, temperature, and the presence of inhibitors. 53 Factors That Affect Enzyme Activity 1- Substrate Concentration Recall that the first step in an enzyme-catalyzed reaction is the formation of ES. At a constant concentration of enzyme, an increase in substrate concentration will cause an increase in the enzyme activity up to the point where the enzyme becomes saturated with substrate. Increasing substrate concentration will not affect the rate of the reaction. A condition known as steady state is when an enzyme is operating under maximum activity. 54 27 Lecturer. KHALED HREEBA  6/21/2024 Substrate concentration (S): 1. First rate (V) increase rapidly (most active sites are empty). 2. Then it slows down ( most active sites are full). 3. Finally it reaches Vmax ( full saturation of all active sites). Vmax The max velocity of an enzymatic reaction when binding site is saturated with substrate. Km ( michalis constant) The substrate concentration at which an enzyme catalyzed reaction proceeds at one-half its maximum velocity 56 28 Lecturer. KHALED HREEBA  6/21/2024 2. Enzyme Concentration : At low enzyme concentration there is great competition for the active sites and the rate of reaction is low. As the enzyme concentration increases, there are more active sites and the reaction can proceed at a faster rate. The velocity of the reaction is directly proportional to the concentration of the enzyme. Further increase in the enzyme concentration will not increase the velocity of the reaction i.e. V α [E] within limits. 29 Lecturer. KHALED HREEBA  6/21/2024 3- pH When the enzyme environment is changed by pH, its tertiary structure is disrupted, altering the active site and causing the enzyme’s activity to decrease. Enzymes are most active at a pH known as their optimum pH. Changes in pH will also affect the nature of the amino acid side chains in the active site. The optimum pH for enzymes is based on the location of the enzymes as shown: 59 60 30 Lecturer. KHALED HREEBA  6/21/2024 4- Temperature Enzymes have an optimum temperature at which they are most active. The optimum temperature for most human enzymes is normal body temperature, 37 oC. Above optimum temperature, enzymes lose activity due to disruption of intermolecular forces stabilizing the tertiary structure. At high temperatures, enzymes denature, which modifies the active site. At low temperatures, enzyme activity is low due to a lack of energy for the reaction to occur. Food is stored in a refrigerator or freezer to slow spoilage brought on by enzymes. Boiling contaminated water will destroy enzymes in bacteria that are present in the water. 62 31 Lecturer. KHALED HREEBA  6/21/2024 Summary Initially, an increase in substrate concentration leads to an increase in the rate of an enzyme-catalyzed reaction. As the enzyme molecules become saturated with substrate, this increase in reaction rate levels off. The rate of an enzyme-catalyzed reaction increases with an increase in the concentration of an enzyme. At low temperatures, an increase in temperature increases the rate of an enzyme-catalyzed reaction. At higher temperatures, the protein is denatured, and the rate of the reaction dramatically decreases. An enzyme has an optimum pH range in which it exhibits maximum activity. 32 Lecturer. KHALED HREEBA  6/21/2024 5. Concentration of Reaction Products: A Enzyme B+C If B and C could be removed as fast as they are formed, the reaction would be 100% complete. The velocity of the reaction is inversely proportional to the concentration of the products. 6. Time: By time velocity ↓↓due to ↓ [S] and ↑ [EP] 7- Inhibitors Inhibitors are types of molecules that will cause enzymes to lose activity. Enzyme inhibitors prevent the active site from interacting with substrate to form ES. Some inhibitors cause temporary loss of activity, while others cause permanent loss of activity. Reversible inhibition occurs when the inhibitor causes a temporary loss of activity. However, activity is regained if the inhibitor is removed. 66 33 Lecturer. KHALED HREEBA  6/21/2024 Reversible inhibitors can be competitive or noncompetitive. ❑ Competitive inhibitors are molecules that compete with a substrate for the active site, and have a structure similar to the substrate. Thus when the substrate and the inhibitor are present, they compete for the active centre of the enzyme. The binding of the inhibitor to the enzyme will decrease the free enzyme available to act on the substrate and the reaction velocity will decrease. As long as an inhibitor remains in the active site, the enzyme cannot react with the substrate to form product. 68 34 Lecturer. KHALED HREEBA  6/21/2024 a. Succinct dehydrogenase enzyme is inhibited by malonic acid which is similar to its substrate succinic acid. CH2 -COOH COOH l CH2 CH2 -COOH COOH Succinic acid Malonic acid b. Sulfanilamide is similar in structure to para-amino benzoic acid used by microorganisms for folic acid synthesis which is vital for their growth, so sulfanilamide is bacteriostatic. c. Dicumarol is similar in structure to vitamin K, so it is used to prevent blood clotting. ❑Noncompetitive inhibitors do not resemble the substrate. They do not compete for the enzyme’s active site. Noncompetitive inhibitors bind at a site on the enzyme that is usually remote to the active site. When a noncompetitive inhibitor binds to an enzyme, it causes a conformational change in the enzyme. This change in shape causes the active site to no longer interact with the substrate. Here there is no similarity in structure between the substrate and inhibitor, so there is no competition between them for the catalytic or binding site of the enzyme. They bind with site other than active site. 70 35 Lecturer. KHALED HREEBA  6/21/2024 This figure diagrams how a noncompetitive inhibitor functions. 71 Non-competitive inhibitors include: A- Allosteric modifiers: These are small organic molecules which have a physiologic regulatory role on enzyme activity. They unite with a locus on the enzyme surface called “the allosteric site”, resulting in conformational changes in the enzyme protein that make it either more suitable to unite with the substrate (allosteric activator) or less suitable to unite with the substrate (allosteric inhibitor). 36 Lecturer. KHALED HREEBA  6/21/2024 The inhibitor is not similar in structure to the substrate and it is bound to the apoenzyme at sites far from the active site. This inhibition is reversible and depends upon the concentration of inhibitor only and not on the substrate concentration. ATP and citrate are allosteric inhibitors for phosphofructokinase enzyme, and glucose-6-phosphate is allosteric inhibitor for hexokinase enzyme. On the other hand, ADP and AMP are allosteric activators to phosphofructokinase. b. Feed back inhibition: Occurs when the end product of sequences of biosynthetic reactions inhibits the activity of an early enzyme. An example of this can be represented by the following reaction, where the compound A is converted to D through intermediate formation of B and C. A Enzyme 1 B Enzyme 2 C Enzyme 3 D A high concentration of D will inhibit enzyme 1 and inhibits conversion of A to B. This is a regulatory process for synthesis of D. 37 Lecturer. KHALED HREEBA  6/21/2024 C. Inhibitors blocking activators and coenzymes: 1. Agents that block the coenzyme will stop enzyme action. Phenylhydrazine will block the aldehyde group of pyridoxal phosphate, which is the coenzyme of transaminases and certain decarboxylases, stopping their action. 2. Agents that block the prostatic group will stop enzyme action. Cyanide and carbon monoxide block the iron of heme of cytochrome oxidase enzyme. Factors That Affect Enzyme Activity, Continued Inhibition by competitive inhibitors can be reversed by adding more substrate. The higher the concentration of substrate, the more likely it will overcome the competition for the active site. Adding more substrate with noncompetitive inhibitors has no effect on overcoming inhibition. Reversing a noncompetitive inhibitor requires a special chemical reagent to remove the inhibitor and restore catalytic activity. 76 38 Lecturer. KHALED HREEBA  6/21/2024 Factors That Affect Enzyme Activity, Continued An irreversible inhibitor forms a covalent bond with an amino acid side chain in the enzyme’s active site. Irreversible inhibition causes the substrate to be excluded from the active site. Irreversible inhibition is a permanent inhibition. 77 Irreversible inhibition is demonstrated in this figure. Heavy metals like silver, mercury, and lead are examples of irreversible inhibitors. 78 39 Lecturer. KHALED HREEBA  6/21/2024 Antibiotics Inhibit Bacterial Enzymes Enzyme inhibitors are used to fight bacterial infections. Penicillin is an example of an irreversible inhibitor. It binds to the enzyme that bacteria use to synthesize cell walls, and slows the growth of cell walls. Without a cell wall, bacteria cannot survive and the infection stops. 79 Enzyme Kinetics Basics Enzyme kinetics studies the reaction rates of enzyme-catalyzed reactions and how the rates are affected by changes in experimental conditions An essential feature of enzyme-catalyzed reactions is saturation: at increasing concentrations of substrates the rate increases and approaches a limit where there is no dependence of rate on concentration Conformation of proteins and positions of side chains are important for enzyme-substrate interactions and catalysis. Forces involved in protein folding and structure are also involved in catalysis- enzyme-substrate specificity 40 Lecturer. KHALED HREEBA  6/21/2024 To use enzymes in biotechnology NEED TO KNOW KINETIC PARAMETERS OF THE ENZYME REACTION. We may want enzymes that WORK FAST- convert more substrate in a fixed unit of time. To do this optimization we have to perform and analyze the enzyme catalyzed reaction. You can adjust pH, temperature and add co-factors to optimize enzyme activity. Like chemical reactions, enzyme catalyzed reactions have kinetics and rates Reaction kinetics is Michaelis-Menten kinetics. Initial Velocity (vo) and [S] The concentration of substrate [S] present will greatly influence the rate of product formation, termed the velocity (v) of a reaction. Studying the effects of [S] on the velocity of a reaction is complicated by the reversibility of enzyme reactions, e.g. conversion of product back to substrate. To overcome this problem, the use of initial velocity (vo) measurements. At the start of a reaction, [S] is in large excess of [P], thus the initial velocity of the reaction will be dependent on substrate concentration 41 Lecturer. KHALED HREEBA  6/21/2024 Substrate Saturation of an Enzyme A. Low [S] B. 50% [S] or Km C. High, saturating [S] Steady State Assumption The M-M equation was derived in part by making several assumptions. An important one was: the concentration of substrate must be much greater than the enzyme concentration. In the situation where [S] >> [E] and at initial velocity rates, it is assumed that the changes in the concentration of the intermediate ES complex are very small over time (vo). This condition is termed a steady-state rate, and is referred to as steady-state kinetics. Therefore, it follows that the rate of ES formation will be equal to the rate ES breakdown. 42 Lecturer. KHALED HREEBA  6/21/2024 Michaelis-Menten equation The Michaelis-Menten equation describes how reaction velocity varies with substrate concentration: V0 = Vmax [S] [S]+Km where Vo = initial reaction velocity Vmax = maximal velocity Km = Michaelis constant = (k-1 + k2)/k1 [S] = substrate concentration The following assumptions are made in deriving the Michaelis-Menten rate equation 43 Lecturer. KHALED HREEBA  6/21/2024 Important Conclusions of Michaels - Menten Kinetics when [S]= KM, the equation reduces to when [S] >> KM, the equation reduces to when [S]