AIU Medical Biochemistry 2024-2025 Lecture Notes PDF

Medical Biochemistry 2024-2025 ENZYMES 1 (Week 8) ILOs: Describe the role of enzymes as biological catalysts Define terms used in enzymology Explain enzyme specificity Define Ribozymes and recognize their functions Introduction to Enzymatic r...

Medical Biochemistry 2024-2025 ENZYMES 1 (Week 8) ILOs: Describe the role of enzymes as biological catalysts Define terms used in enzymology Explain enzyme specificity Define Ribozymes and recognize their functions Introduction to Enzymatic reactions Enzymes are specialized proteins that function in the acceleration of chemical reactions. Many reactions required for normal activity of cells would not proceed fast enough at the pH and temperature of the body without these specialized proteins. The term defining the speed of a chemical reaction, whether catalyzed or uncatalyzed, is rate or velocity. Rate (velocity) is the change in amount of starting materials (substrates) or products per unit time. Enzymes increase the rate by acting as catalysts. A catalyst increases the rate of a chemical reaction but is not itself changed in the process. Two important characteristics of enzyme catalysts are that the enzyme is not changed as a result of catalysis and the enzyme does not change the equilibrium constant of the reaction but simply increases the rate at which the reaction approaches equilibrium. Enzymes may be considered to lower energy barriers for chemical reactions i.e., reduce activation energy. 1  Medical Biochemistry 2024-2025  Transition state Unstable arrangement of atoms in which chemical bonds are in the process of being formed or broken.  Activation energy The energy required to reach the transition state from the ground state of the reactants. 2  Medical Biochemistry 2024-2025 Enzymes as biological catalysts ✓ Enzymes are almost all very large globular proteins of high molecular weight. ✓ Act at very small concentrations. ✓ Increase the speed of chemical reactions without changing the result. ✓ At the end of the reaction the structure of the enzyme is unchanged ✓ Thermolabile, (substance that is subject to destruction /decomposition/denaturation in response to heat) so lose their activity when the body temperature is high. !! RIBOZYMES: Non-protein Organic catalysts ✓ Segments of RNA ✓ Display an enzyme activity in the absence of protein ✓ Acting on themselves or other RNA molecules ✓ Require divalent metal cations (eg. Mg2+) as cofactors Terms used in enzymology Substrate: Molecule that enters a reaction to be transformed by the catalytic activity of an enzyme. All molecules that enter an enzymatic reaction and are permanently modified are called substrates. Product: The substance that is produced by the action of the enzyme. 3  Medical Biochemistry 2024-2025 Apoenzyme: protein portion of an enzyme which is catalytically inactive. Coenzyme: Essential for the activity of enzymes and they may be considered as second substrates or Co-substrate. Catalytic site: a particular arrangement of amino acids side chain in the polypeptide, specific to bind a specific substrate, also contains the machinery involved in catalyzing the reaction. Allosteric site: a region of enzyme molecules not at the catalytic site where small molecules bind and effect a change in the activity of the active site by change in the conformation of the enzyme. This causes the active site to become either more active or less active by increasing or decreasing the affinity of enzyme for substrate. Active site : A more general term which involves all sequences of amino acids which affect the activity of the enzyme. Includes for example the catalytic site, the allosteric sites Naming (Nomenclature) of Enzymes A suffix (ase) is added to indicate enzyme. The prefix may be: 1. Name indicating general nature of substrate e.g.: protease, lipase etc..... 2. Real name of substrate: urease, lactase, sucrase. 3. Type of reaction catalyzed e.g.: Aldolase, mutase, transaminase, dehydrogenase etc... 4  Medical Biochemistry 2024-2025 4. Combination e.g.: Name of substrate + type of reaction Lactic acid Dehydrogenase Name of product + type of reaction Glycogen Synthetase Enzyme Specificity Enzymes show different levels of specificity: 1) Absolute specificity The enzyme acts only on a specific substrate e.g., Lactase acting on lactose. 2) Relative specificity The enzyme acts at different rates on one type of bond in compounds chemically related, e.g., pancreatic lipase hydrolyzes alpha ester bonds at position 1 & 3 in Triglycerides, (whether these are tristearin, triolein, tripalmitin). 3) Group specificity (structural specificity) The enzyme acts on a special type of bond at specific site and attached to specific groups. e.g., Pepsin that acts on peptide bond between amino group of an aromatic amino acid and carboxylic group of another amino acid. While Trypsin acts on peptide bond between carboxylic group of basic amino acid and amino group of another amino acid. 5  Medical Biochemistry 2024-2025 Figure: Pepsin activity demonstrating group specificity. 4) Stereochemical specificity D- amino acid oxidase acts only on D- amino acid. L- amino acid oxidase acts only on L- amino acids. Figure: L-Alanine oxidase activity demonstrating stereochemical specificity. 6  Medical Biochemistry 2024-2025 Functional vs Non-functional plasma enzymes Functional plasma enzymes Enzymes present in the plasma at high concentrations all the time because they have functional role, examples include: ✓ Thrombin is associated with blood coagulation. ✓ Plasmin is associated with fibrin dissolution. Non-functional plasma enzymes Enzymes present at very low levels in healthy individuals and play no functional role in plasma. They result from normal turnover of cells. In disease, levels of these non- functional enzymes increase in plasma due to: ** Change in cell membrane permeability ** Increased cell death Intracellular enzymes of low molecular weight appear in plasma first. Cytosolic enzymes appear in plasma before mitochondrial enzymes. The greater the quantity of tissue damaged, the more the increase in plasma level. Role of enzymes in Diagnosis of diseases: Some enzyme levels are increased in plasma in some diseases: Alkaline phosphatase in obstructive jaundice Creatine kinase in heart diseases Acid phosphatase in prostatic carcinoma 7  Medical Biochemistry 2024-2025 Role of enzymes in Treatment of diseases: Some enzymes are used in the treatment of some diseases. Examples include: Streptokinase in treatment of infarction. Digestive enzymes in the treatment of maldigestion. ENZYMES 2 (Week 9) ILOs: Outline the process of substrate binding and enzyme action Discuss different factors affecting enzyme activity Enumerate different mechanisms of enzyme activation Compare competitive and non-competitive enzyme inhibitors Discuss allosteric modifiers Outline different methods of enzyme regulation Define isoenzymes and outline their use Classify enzymes based on type of reaction Substrate binding and enzyme action Enzyme and substrate combine to form a complex E + S ↔ ES 8  Medical Biochemistry 2024-2025 Complex goes through a transition state – not quite a substrate or product ES ↔ ES* A complex of the enzyme and the product is produced ES* ↔ EP. Finally, the enzyme and product separate EP ↔ E + P Characteristics of the active site of the enzyme Enzymes are the most specific catalysts known, as regards the substrate and the type of reaction undergone. Specificity resides in the substrate binding site on the enzyme surface (catalytic site). The tertiary structure of the enzyme is folded in such a way as to create a region that has the correct molecular dimensions and the optimal alignment of counter ionic groups and hydrophobic regions to accommodate a specific substrate. Enzymes use weak, mostly non-covalent interactions to hold the substrate in place based on R groups of amino acids. Shape is complementary to the substrate and determines the specificity of the enzyme. 9  Medical Biochemistry 2024-2025 Complementarity of Substrate and Enzyme Explains Substrate Specificity Various models have been proposed to explain the substrate specificity of enzymes. I- Lock and key model: This model assumes that only a substrate of the proper shape could fit with the enzyme. Figure: Lock and key model. II- Induced fit model: A more flexible model of the binding site is provided by the induced fit model in which the binding and active sites are not fully preformed. The essential elements of the binding site are present to the extent that the correct substrate can position itself properly. Interaction of substrate with enzyme induces a conformational change in the enzyme, resulting in the formation of 10  Medical Biochemistry 2024-2025 a stronger binding site and the repositioning of the appropriate amino acids to form the active site. Factors affecting Enzyme activity Measurement of the rate of enzyme reaction (velocity) Rate of change of substrate to product per unit of time. Vmax is the maximum reaction velocity at which all enzymes become saturated with substrate Enzyme activity can be influenced by: 1) Substrate concentration 2) Enzyme Concentration 3) Coenzyme concentration 4) Product concentration 5) pH 11  Medical Biochemistry 2024-2025 6) Temperature 7) Physical agents 8) Time 9) Enzyme inhibitors 10) Enzyme activators 1) Effect of substrate concentration on Reaction Velocity: ▪ The first part of the curve is called the first order of enzyme activity where an increase in substrate concentration increases the rate of the enzyme reaction till Vmax. The second part of the curve is called zero order of enzyme activity after the Vmax where there is no further increase in reaction velocity with increased substrate concentration. i.e., The substrate is in excess. This means all substrate binding sites of the enzyme are occupied by the substrate. ▪ Km (The Michaelis–Menten constant of an enzyme) is the substrate concentration at which the enzyme works at half its maximum rate. (It is used as a measure of the affinity of the enzyme for its substrate) The higher the affinity of an enzyme for its substrate, the lower the substrate concentration needed till ½Vmax is reached. The lower the substrate concentration, the lower the value of Km. So the higher the affinity of an enzyme for its substrate, the lower its Km will be. It is a constant for an enzyme, but it differs from enzyme to another. ✓ Small Km means tight binding- high affinity. 12  Medical Biochemistry 2024-2025 ✓ High Km means weak binding- low affinity. 1st order Zero order Figure: Effect of substrate concentration. 2) Effect of enzyme concentration: Increase in enzyme concentration increases the rate of reaction (1st order) till a certain point where the increase in enzyme concentration does not increase 13  Medical Biochemistry 2024-2025 the rate of reaction (zero order) as the substrate is completely combined with the enzyme. Figure: Effect of enzyme concentration. 3) Effect of Coenzyme concentration on the enzymatic activity Coenzyme concentration has the same effect and gives the same curve of substrate concentration on enzymatic activity. 4) Effect of Product concentration on Enzymatic activity Increased product concentration decreases the enzyme activity due to: a. Change in the pH. b. The product may be more or less similar to the substrate so it may compete with it for binding on its active site. c. The product may bind the enzyme at an allosteric site. 5) Effects of pH on enzyme activity 14  Medical Biochemistry 2024-2025 ▪ Each enzyme has an optimum pH at which appropriate charges are present on both S and E. ▪ Below and above this optimum pH, the enzyme activity is diminished. ▪ At the extremes of pH the enzyme activity is lost due to denaturation. 6) Effect of Temperature on enzyme activity ▪ Optimum Temp. is temp. at which the enzyme reaction velocity is maximal. ▪ It is usually 25-40ºC in the human body. ▪ Exceeding normal temperature ranges always reduces enzyme reaction rate. ▪ An increase in temperature above 45ºC decreases the enzymatic activity irreversibly due to enzyme denaturation. Figure: Effect of pH & temperature on enzyme activity. 15  Medical Biochemistry 2024-2025 7) Effect of Physical agents on enzymatic activity Decrease in Enzyme activity Increase in Enzyme activity Heating Red light Shaking Blue light Stirring Infrared light Ultraviolet light Importance of enzyme inhibition studies The active site of an enzyme fits its substrate perfectly. It is possible, however, for a molecule which is similar in shape to the substrate (analog) to enter an enzyme’s active site. This would then inhibit the enzyme’s function. Many substances can inhibit enzyme activity as substrate analog, toxins, drugs, metal complexes. Inhibition studies can provide: Better understanding of enzyme reaction mechanisms. Information on metabolic pathways. Insight on how drugs and toxins exert their effects. 16  Medical Biochemistry 2024-2025 Enzyme inhibitors Two broad classes of inhibitors have been identified based on the extent of inhibition: Reversible: Forms weak, non-covalent bonds that readily dissociate from an enzyme. The enzyme is only inhibited when the inhibitor is present. Irreversible (enzyme poison): Forms covalent or very strong non-covalent bonds. The site of attack is an amino acid group that participates in the normal enzymatic reaction. Types of Reversible Inhibitors a) Competitive inhibitors The enzyme can bind substrate forming (ES) complex, or inhibitor forming (EI) but not both. Competitive inhibitor (I) binds only to E, not to ES. Competitive inhibitors resemble the substrate and bind to the active site of the enzyme. The substrate is therefore prevented from binding to the same active site. 17  Medical Biochemistry 2024-2025 A competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate. Competitive inhibition can be overcome by increasing the concentration of substrate. Kinetically in competitive inhibition Vmax is not altered, while Km is increased as the affinity of E to S decreased. b) Non-competitive inhibition (Allosteric) The inhibitor and substrate can bind simultaneously to an enzyme molecule. Hence their binding sites do not overlap. Non-competitive inhibitor (I) binds either to E and/or to ES. While the inhibitor is bound to the enzyme, it can seriously affect the normal arrangement of hydrogen bonds and hydrophobic interactions holding the enzyme molecule in its three-dimensional shape. The resulting distortion changes the shape of the active site and therefore inhibits the ability of the substrate to enter the active site. While the inhibitor is attached to the enzyme, the enzyme’s function is blocked. Non-competitive inhibition cannot be overcome by increasing the substrate concentration. ▪ Kinetically Vmax is decreased, while Km is not affected. 18  Medical Biochemistry 2024-2025 Inhibition of an enzyme can be harmful or even fatal but, in many situations, inhibition is essential. For example, metabolic reactions must be controlled so that no enzyme can be allowed to work without stopping at some point. One way of controlling metabolic reactions is to use the end product of a chain of reactions as a non-competitive, reversible inhibitor (End product or Feed back inhibition). Figure: End product allosteric inhibition, Enzyme Activators The velocity of the enzymatic reaction is affected by the concentration of an enzymatic activator. Some enzymes are activated by different ways: 1) Allosteric activation. 2) Covalent modification, e.g., phosphorylation and dephosphorylation. 3) Removal of an inhibitory peptide converts a zymogen (or proenzyme) into an active enzyme: 19  Medical Biochemistry 2024-2025 e.g., Trypsinogen Trypsin + inhibitory peptide *Proenzyme (Zymogen): inactive precursor form of some enzymes (e.g. many digestive enzymes) that will be activated by cleavage of a specific peptide in its structure. 4) Keeping -SH group in an active site of some enzymes in its reduced form by a reducing agent such as vitamin C. 5) Some enzymes need a metal ion to be active. Key Regulatory Enzymes The thousands of reactions involved in metabolism are grouped in sequence. There is a step or more involve regulatory enzymes that control the rate for the entire sequence. Committed-Step Enzyme The first irreversible enzyme unique to the pathway. Rate-limiting Enzyme: The enzyme with the lowest Vmax. Usually occurs early in the pathway. Enzyme activity is regulated in multiple ways:  Long term- Slow (regulating the amount) through synthesis &/or degradation.  Short term- Rapid (regulating the activity).  Compartmentation Enzyme Regulation by Changing Enzyme amount (Quantitative) 20  Medical Biochemistry 2024-2025 a) Induction Increase in the synthesis of the enzyme by the effect of an inducer, which may be a hormone or a metabolite. Enzymes subject to induction (Inducible Enzymes) are those needed at only one stage of development, or under selected physiological conditions. Enzymes that are in constant use are not regulated by altering the rate of synthesis and are called Constitutive Enzymes. b) Repression Inhibition of enzyme synthesis by a repressor, which may be a hormone or a metabolite. c) Enzyme degradation Individual enzyme molecules have different lifespans. Some enzymes last for many days, others for only minutes or less. Enzymes at key control points are more rapidly degraded. Enzyme Regulation by Compartmentation The activity of the pathway can be regulated by physical partitioning of the pathway from its initial substrate. This controls the access of the substrate to the enzyme of the pathway. Anabolic and catabolic pathways are usually segregated into different organelles in the cell to maximize the cellular economy. Most synthetic enzymes are present extra- mitochondrial. 21  Medical Biochemistry 2024-2025 Most of the oxidative (degradative) enzymes are present in the mitochondria. Isoenzymes Enzymes that differ somewhat in primary structure and properties from tissue to tissue, but retain essentially the same function, are called tissue-specific isoforms or isoenzymes. Act on the same substrate and give the same product. Have quaternary structure. Differ in physical and chemical properties, so are different in electrophoretic mobility. They differ in kinetics: different Km and Vmax values; differ in their affinity for substrates. Since present in different tissues, they are used in diagnosis of the exact site of their release in case of elevation of the enzyme. Example of clinically important isoenzymes: I- Lactate dehydrogenase (LDH) ▪ The best isoenzymes known which catalyze the conversion of pyruvate to lactate. ▪ It is a Tetrameric enzyme containing two different subunits designated H (for heart) and M (for muscle). ▪ These subunits are made by separate genes. 22  Medical Biochemistry 2024-2025 ▪ The subunits have slightly different amino acid sequences and so have different catalytic properties. ▪ The subunits associate to form tetramers randomly. ▪ A total of five different isoenzymes of LDH are found in the body. Type of Structure Organs isoenzyme LDH-1 HHHH (H4) 1. Cardiac muscle LDH-2 HHHM (H3M) 1. Reticuloendothelial LDH-3 HHMM(H2M2) 1. Mainly lung tissue 2. Pancreas 3. Spleen 4. Lymphocytes LDH-4 HMMM (HM3) 1. Kidney 2. Pancreas 3. Intestine LDH-5 MMMM (M4) 1. Liver 2. Skeletal muscles II- Creatine kinase (CK) ▪ Dimer with two types of subunits, M (muscle type) and B (brain type) ▪ In brain, BB (CK1) ▪ In heart, MB (CK2) ▪ In muscle, MM (CK3) ▪ In other tissues variable amounts of MM and BB are present 23  Medical Biochemistry 2024-2025 International Classification of Enzymes No. Class Type of reaction catalyzed 1 Oxidoreductases Transfer of electrons 2 Transferases Group transfer reactions (not hydrogen) 3 Hydrolases Hydrolysis reactions (bond cleavage by water introduction & transfer of functional groups to water) 4 Lyases Addition of groups to double bonds, or formation of double bonds by removal of groups (break C-O, C-C or C-N bonds without using water) 5 Isomerases Rearrangement of groups within molecules to yield isomeric forms. ** enzymes catalyzing movement of a phosphate from one atom to another are called mutases 6 Ligases Formation of C-C, C-S, C-O, and C-N bonds by condensation reactions coupled to ATP cleavage 24  Medical Biochemistry 2024-2025 25