Biochemistry: Enzymology (SY 2024-2025) PDF

BIOCHEMISTRY: MELVIN G. BERIN, MD SY 2024-2025 References: 1. Harper’s Illustrated Biochemistry 2. Textbook of Biochemistry for Medical Students by Vasudevan 3. https://www.khanacademy.org/science/ap-biology/cellular-energetics/environmental-impacts-on-enzyme-func...

BIOCHEMISTRY: MELVIN G. BERIN, MD SY 2024-2025 References: 1. Harper’s Illustrated Biochemistry 2. Textbook of Biochemistry for Medical Students by Vasudevan 3. https://www.khanacademy.org/science/ap-biology/cellular-energetics/environmental-impacts-on-enzyme-function/a/enzyme-regulation Act as biological catalysts (biocatalysts) The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. Metabolic pathways depend upon enzymes to catalyze individual steps CHARACTERISTICS i. Almost all enzymes are proteins. Enzymes follow the physical and chemical reactions of proteins. Notable exceptions include ribosomal RNAs and a handful of RNA molecules known collectively as ribozymes ii. are heat labile. iii. are water-soluble. iv. can be precipitated by protein precipitating reagents (ammonium sulfate or trichloroacetic acid). v. contain 16% weight as nitrogen. CLASSIFICATION Some of the names for enzymes first described in the earliest days of biochemistry persist in use to this day. Examples include pepsin, trypsin, and amylase. However, in most cases early biochemists designated newly discovered enzymes by first appending the suffix –ase to a descriptor for the type of reaction catalyzed. For example, enzymes that remove hydrogen atoms are generally referred to as dehydrogenases, enzymes that hydrolyze proteins as proteases, and enzymes that catalyze rearrangements in configuration as iso merases IUBMB System of Classification o Class 1. Oxidoreductases: ▪ Transfer of hydrogen or addition of oxygen ▪ This group of enzymes will catalyze oxidation of one substrate with simultaneous reduction of another substrate or co-enzyme. This may be represented as AH2 + B -------------→ A + BH2 for example, Alcohol + NAD+ ----→ Aldehyde + NADH + H+ The enzyme is Alcohol dehydrogenase; IUB name is Alcohol-NAD-oxidoreductase ▪ e.g. Lactate dehydrogenase; Glucose-6-phosphate dehydrogenase; Succinate dehydrogenase o Class 2. Transferases: ▪ Transfer of groups other than hydrogen.  ▪ This may be represented as A-R + B → A + B-R For example, Hexose + ATP → Hexose-6-phosphate + ADP The name of enzyme is Hexokinase and IUB name is ATP-Hexose--6-phosphate transferase. ▪ e.g Aminotransferase o Class 3. Hydrolases: ▪ This class of enzymes can hydrolyze ester, ether, peptide or glycosidic bonds by adding water and then breaking the bond. Acetyl choline + H2O --------→ Choline + acetate The enzyme is Acetyl choline esterase or Acetyl choline hydrolase (IUB name) ▪ All digestive enzymes are hydrolases ▪ e.g. Acetyl choline esterase; Trypsin o Class 4. Lyases: ▪ Cleave without adding water ▪ These enzymes can remove groups from substrates or break bonds by mechanisms other than hydrolysis. For example, Fructose-1,6-bisphosphate -------→ Glyceraldehyde-3-phosphate +dihydroxy acetone phosphate The enzyme is Aldolase ▪ e.g. HMG CoA lyase; ATP Citrate lyase. o Class 5. Isomerases: ▪ Intramolecular transfers. ▪ These enzymes can produce optical, geometric or positional isomers of substrates. ▪ e.g. Racemases, epimerases, cis-trans isomerases o Class 6. Ligases: ▪ ATP dependent condensation of two molecules, ▪ These enzymes link two substrates together, usually with the simultaneous hydrolysis of ATP, (Latin, Ligare = to bind). For example, Acetyl CoA + CO2 + ATP → Malonyl CoA + ADP + Pi Enzyme is Acetyl CoA carboxylase. ▪ e.g. Glutamine synthetase; PRPP synthetase Synthetase and Synthase are Different o Synthetases are ATP-dependent enzymes catalyzing biosynthetic reactions; they belong to Ligases (class 6). Examples are Carbamoyl phosphate synthetase; Arginine succinate synthetase; PRPP synthetase and Glutamine synthetase o Synthases are enzymes catalyzing biosynthetic reactions; but they do not require ATP directly; they belong to classes other than Ligases. Examples are Glycogen synthase and ALA synthase CLINICAL CORRELATES: Thousands of diseases related to deficient or defective enzymes occur. Examples: 1. Phenylketonuria (PKU) (which has an incidence of 1 in 10.000 births in whites and Asians), the enzyme phenylalanine hydroxylase, which converts phenylalanine to tyrosine, is deficient Phenylalanine accumulates, and tyrosine becomes an essential amino acid that is required in the diet Intellectual disability is a result of this metabolic derangement. in part as a result of the brain lacking various essential amino acids because of the elevated levels of phenylalanine in the circulation. 2. A more common problem is lactase deficiency, which occurs in more than 80% of Native Americans, African Americans, and Asian Americans. Lactose is not digested at a normal rate and accumulates in the gut where it is metabolized by bacteria. Bloating, abdominal cramps, and watery diarrhea result. 3. Emphysema may result from an inherited deficiency of α₁-antitrypsin, an enzyme that inhibits elastase action in the lungs. Elastase is a serine protease found in neutrophils that utilize the enzyme to destroy inhaled organisms in the air. At times, the elastase may escape from the neutrophil and then the protease begins to destroy the lung cells. The circulating protein cx1-antitrypsin blocks the action of elastase and protects the lung from damage. Cigarette smoke contains oxidizing agents that will destroy a key methionine residue in α₁-1-antitrypsin and destroys α₁--antitrypsin activity. Smoking for an extended period of time increases the chances of developing emphysema. CO-ENZYME Enzymes may be simple proteins, or complex enzymes, containing a non-protein part, called the prosthetic group. The prosthetic group is called the co-enzyme. It is heat stable. The protein part of the enzyme is then named the apo-enzyme. It is heat labile. These two portions combined together is called the holo-enzyme. Salient Features of Co-enzymes 1. The co-enzyme is essential for the biological activity of the enzyme. 2. Co-enzyme is a low molecular weight organic substance. It is heat stable. 3. Generally, the co-enzymes combine loosely with the enzyme molecules. The enzyme and coenzyme can be separated easily by dialysis. 4. Inside the body, when the reaction is completed, the co-enzyme is released from the apo-enzyme, and can bind to another enzyme molecule. 5. One molecule of the co-enzyme is able to convert a large number of substrate molecules with the help of the enzyme. 6. Most of the co-enzymes are derivatives of vitamin B complex group of substances  Co-enzymes may be divided into two groups 1. First Group of Co-enzymes o Those taking part in reactions catalyzed by oxidoreductases by donating or accepting hydrogen atoms or electrons. o the change occurring in the substrate is counter-balanced by the co-enzymes. Therefore, such co-enzymes may be considered as co-substrates or secondary substrates o In the example shown, the substrate lactate is oxidized, and simultaneously the co -enzyme (co- substrate) is reduced. If the reaction is reversed, the opposite effect will take place o o Other such examples are NADP–NADPH; FAD-FADH2 and FMN–FMNH2. nd 2. 2 Group of Co-enzymes o These co-enzymes take part in reactions transferring groups other than hydrogen. A particular group or radical is transferred from the substrate to another substrate. Most of them belong to vitamin B complex group. A few such examples are o ATPAdenosine Triphosphate (ATP) ▪ ATP is considered to be the energy currency in the body. ▪ In the ATP molecule, the second and third phosphate bonds are ‘high energy' bonds (as shown with squiggle bonds in Figure ▪ During the oxidation of food stuffs, energy is released, a part of which is stored as chemical energy in the form of ATP. METALLO-ENZYMES These are enzymes which require certain metal ions for their activity. Some examples are given in Table. In certain cases, e.g. copper in Tyrosinase, the metal is tightly bound with the enzyme. In other cases, even without the metal ion, enzyme may be active; but when the metal ion is added, the activity is enhanced. They are called ion-activated enzymes, e.g. calcium ions will activate pancreatic lipase MODE OF ACTION OF ENZYMES 1. Lowering of Activation Energy o Enzymes lower the energy of activation. o Activation energy is defined as the energy required to convert all molecules of a reacting substance from the ground state to the transition state  2. Acid Base Catalysis o Many acid-base catalysis reactions involve histidine because it has a pH close to 7, allowing it to act as both an acid and a base with the help of ribonuclease 3. Substrate Strain o Binding of substrate to a preformed site on the enzyme can induce strain in the substrate. The energy level of the substrate is raised. A combination of substrate strain and acid base catalysis is seen in the action of lysozyme. 4. Serine proteases o enzymes that cleave peptide bonds in proteins o e.g. trypsin, chymotrypsin, clotting factors 5. Covalent catalysis o involves the substrate forming a transient covalent bond with residues in the enzyme active site or with a cofactor. This adds an additional covalent intermediate to the reaction, and helps to reduce the energy of later transition states of the reaction. 6. Entropy effect o Enzymes enhance reaction rates by decreasing entropy o reduces disorder by orienting substrates for reaction PRODUCT SUBSTRATE ORIENTATION THEORIES  A. MICHAELIS–MENTEN THEORY ▪ Enzyme–Substrate complex theory. ▪ Accordingly, the enzyme (E) combines with the substrate (S), to form an enzyme-substrate (ES) complex, which immediately breaks down to the enzyme and the product (P) ▪ E + S ↔ E–S Complex → E + P B. FISCHER'S TEMPLATE THEORY ▪ It states that the 3D of the active site of the enzyme is complementary to the substrate. Thus, enzyme and substrate fit each other, similar to lock and key. The lock can be opened by its own key only ▪ However, Fischer envisaged a rigid structure for enzymes, which could not explain the flexibility shown by enzymes C. KOSHLAND'S INDUCED FIT THEORY ▪ Conformational changes are occurring at the active site of enzymes concomitant with the combination of enzyme with the substrate ACTIVE SITE OR ACTIVE CENTER OF ENZYME o The region of an enzyme where substrate molecules bind and undergo a chemical reaction. o The active site consists of amino acid residues that form temporary bonds with the substrate (binding site) and residues that catalyze a reaction of that substrate (catalytic site). THERMODYNAMIC CONSIDERATIONS From the standpoint of energy, the enzymatic reactions are divided into 3 types: 1. Exergonic or Exothermic Reaction ▪ Here energy is released from the reaction, and therefore reaction essentially goes to completion, ▪ e.g. urease enzyme: Urea -----------→ ammonia + CO2 + energy ▪ At equilibrium of this reaction, the substrate will be only 0.5% and product will be 99.5%. ▪ Such reactions are generally irreversible. 2. Isothermic Reaction ▪ When energy exchange is negligible, and the reaction is easily reversible, ▪ e.g. Pyruvate + 2H ↔ Lactate 3. Endergonic or endothermic ▪ Energy is consumed and external energy is to be supplied for these reactions. ▪ In the body this is usually accomplished by coupling the endergonic reaction with an exergonic reaction, e.g. Hexokinase catalyses the following reaction: Glucose + ATP → Glucose-6-phosphate + ADP FACTORS INFLUENCING ENZYME ACTIVITY 1. Enzyme Concentration Rate of a reaction or velocity (V) is directly proportional to the enzyme concentration, when sufficient substrate is present. Velocity of reaction is increased proportionately with the concentration of enzyme, provided substrate concentration is unlimited 2. Effect of Substrate Concentration As substrate concentration is increased, the velocity is also correspondingly increased in the initial phases; but the curve flattens afterwards Further increase in substrate cannot make any effect. The maximum velocity obtained is called Vmax. It represents the maximum reaction rate attainable in presence of excess substrate. Km (Michaelis constant) is the concentration of substrate which permits the enzyme to achieve half Vmax. Km is the Signature of the Enzyme. Km value is thus a constant for an enzyme. It is the characteristic feature of a particular enzyme for a specific substrate 3. Effect of Concentration of Products In a reversible reaction, S ↔ P, when equilibrium is reached, as per the law of mass action, the reaction rate is slowed down. So, when product concentration is increased, the reaction is slowed, stopped or even reversed. In inborn errors of metabolism, one enzyme of a metabolic pathway is blocked. For example: E1 E2 E3 A -------→ B ----------→ C -------||------→ D If E3 enzyme is absent, C will accumulate, which in turn, will inhibit E2. Consequently, in course of time, the whole pathway is blocked. 4. Effect of Temperature The velocity of enzyme reaction increases when temperature of the medium is increased; reaches a maximum and then falls (Bell shaped curve). The temperature at which maximum amount of the substrate is converted to the product per unit time is called the optimum temperature Most human enzymes have the optimum temperature around 37°C. Certain bacteria living in hot springs will have enzymes with optimum temperature near 100°C. 5. Effect of Hydrogen ion concentration (pH) Each enzyme has an optimum pH, on both sides of which the velocity will be drastically reduced. Optimum pH may vary depending on the temperature, concentration of substrate, presence of ions etc. Usually, enzymes have the optimum pH between 6 and 8. Some important exceptions are pepsin (with optimum pH 1-2); alkaline phosphatase (optimum pH 9-10) and acid phosphatase (4-5). 6. Presence of Activators A. In presence of certain inorganic ions, some enzymes show higher activity. Thus, chloride ions activate salivary amylase and calcium activate lipases. B. Another type of activation is the conversion of an inactive pro-enzyme or zymogen to the active enzyme. e.g. By splitting a single peptide bond, and removal of a small polypeptide from trypsinogen, the active trypsin is formed. 7. Presence of inhibitors A. Competitive Inhibition Here inhibitor molecules are competing with the normal substrate molecules for binding to the active site of the enzyme, because the inhibitor is a structural analog of the substrate. Is usually reversible. Or, excess substrate abolishes the inhibition. Km is increased in presence of competitive inhibitor but Vmax is not changed. B. Non-competitive Inhibition (Mostly Irreversible) A variety of poisons, such as lead, cyanide & mercury act as irreversible non-competitive inhibitors. There is no competition between substrate and inhibitor The inhibitor usually binds to a different domain on the enzyme, other than the substrate binding site. Since these inhibitors have no structural resemblance to the substrate, an increase in the substrate concentration generally does not relieve this inhibition. The velocity (Vmax) is reduced but Km value is not changed C. Uncompetitive Inhibition (Can be Reversible or Irreversible) Here inhibitor does not have any affinity for free enzyme. Inhibitor binds to enzyme– substrate complex; but not to the free enzyme. In such cases both Vmax and Km are decreased D. Allosteric Regulation Allosteric Enzyme has one catalytic site where the substrate binds and another separate allosteric site where the modifier binds (allo = other) The binding of the regulatory molecule can either enhance the activity of the enzyme (allosteric activation), or inhibit the activity of the enzyme (allosteric inhibition). In the former case, the regulatory molecule is known as the positive modifier and in the latter case as the negative modifier. Body uses allosteric enzymes for regulating metabolic pathways. Such a regulatory enzyme in a particular pathway is called the key enzyme or rate limiting enzyme. The allosteric inhibitor is most effective when substrate concentration is low. This is metabolically very significant. When more substrate molecules are available, there is less necessity for stringent regulation. An example is given in detail below: This is the first step in heme biosynthesis. The end product, heme will allosterically inhibit the ALA synthase. This enzyme is the key enzyme of heme synthesis.  8. 9. Stabilization Enzyme molecules undergo usual wear and tear and finally get degraded. Such degradation if prevented can lead to increased overall enzyme activity. This is called stabilization of enzyme. e.g 1. Degradation of Tryptophan pyrrolase is retarded by tryptophan. 2. Phospho fructo kinase is stabilized by growth hormone. 3. Enzymes having SH groups (Papain, Urease, Succinate dehydrogenase) are stabilized by glutathione 10. Compartmentalization Certain enzymes of the pathway may be located in mitochondria whereas certain other enzymes of the same pathway are cytoplasmic. CLINICAL CORRELATES: Drugs are frequently used therapeutically to inhibit enzymes; Examples: 1. 5-fluorouracil (5-FU) is used to inhibit the enzyme thymidylate synthase. This enzyme converts deoxy- uridine monophosphate (dUMP) to deoxy-thymidine monophosphate (dTMP), which ultimately provides the thymine for DNA synthesis. 5-FU is used as a chemotherapeutic agent to inhibit the proliferation of cancer cells. 2. Aspirin irreversibly inhibits the enzyme cyclooxygenase and interferes with the generation of prostaglandins and thromboxanes, the secondary mediators of pain signaling. 3. Allopurinol is a suicide inhibitor of xanthine oxidase and is used to treat gout. Xanthine oxidase will recognize allopurinol as a substrate and oxidize it to create oxypurinol, which binds so tightly to the active site that it is not released and then inhibits further reactions by the enzyme. 4. Disulfiram, a drug used to treat alcoholism irreversibly inhibits the enzyme aldehyde dehydroganase, leading to an accumula1ion of acetaldehyde whenever alcohol is ingested. The acetaldehyde leads to “hangover" symptoms and may deter the patient from drinking alcohol.