Enzymes PDF

# Enzymes ## Definition: Organic substances act as a biocatalyst that ↑ the rate of chemical reaction. ## Properties: - Most enzymes are protein in nature (some are RNA in nature called ribozyme). - Neither produced nor consumed in the chemical reactions. - Not changed chemically at the end of t...

# Enzymes ## Definition: Organic substances act as a biocatalyst that ↑ the rate of chemical reaction. ## Properties: - Most enzymes are protein in nature (some are RNA in nature called ribozyme). - Neither produced nor consumed in the chemical reactions. - Not changed chemically at the end of the reaction. - Doesn't affect the equilibrium constant (ΔG is the same). - Highly specific in their action (acts on a specific substrate or few related substrates). - Produced by the living cell (cellular catalyst) but can work in vivo and in vitro. - Needed in very small amounts for the chemical reaction. ## Nomenclature: 1. Old Method: Pepsin - Trypsin - Chymotrypsin - Rennin. 2. New Method: - Hydrolase: Substrate + ase as in sucrase, urease, arginase, and glucosidase. - Other action: Substrate + action of the enzyme as: - Lactate dehydrogenase. - Pyruvate carboxylase. - Adenylate cyclase. ## Enzyme Specificity: **Def:** Enzymes are highly specific in their actions, interacting with 1 (absolute specificity) or few related substrates (relative specificity). **Cause:** Enzyme specificity is due to the nature and arrangement of the chemical groups at the catalytic site. ## Importance of Enzyme Specificity: - Digestive enzymes are of low specificity allowing few numbers of enzymes to digest all food stuffs. - Metabolic enzymes are of high specificity to be well regulated. ## Chemical Nature of Enzymes | Type | Description | |---|---| | Simple protein enzymes | Formed only of amino acids | | Conjugated protein enzymes | Formed from a protein part (apoenzyme) and non-protein part (cofactor) | - Except for ribozymes (RNA in nature) all enzymes are protein in nature. - According to the chemical nature enzymes are classified into simple protein enzymes and conjugated protein enzymes. ## Conjugated Protein Enzyme: - Conjugated protein enzyme is formed of protein part (apoenzyme) and non-protein part (cofactor) both are Called holoenzyme. - Apoenzyme alone is inactive. - Cofactors may be: - Coenzymes: Prosthetic group or Co-substrate - Metal ion: Metalloenzymes or Metal activated enzyme ## Cofactors | Type | Description | |---|---| | Organic | Coenzyme, Prosthetic group | | Inorganic | Co-substrate, Metalloenzyme, Metal activated enzyme | - **Prosthetic Group:** Tightly bound to the apoenzyme by covalent or non-covalent forces - **Co-substrate:** Loosely bound to the apoenzyme - **Metalloenzyme:** Cation is tightly bound to the apoenzyme - **Metal activated enzyme:** Cation loosely bound to apoenzyme ## Mechanism of Enzyme Action - Enzyme is a large protein molecule that contains a small specific region as: - **Active site (catalytic site) or substrate binding site:** complimentary to the substrate - The amino acids are arranged in a precise manner that make the enzyme specific to one substrate or few related substrates. - The active site is rich in many groups as COOH, NH₂, SH and OH. - The active site is rich in certain amino acids as serine, histidine, cysteine, aspartate and glutamate. - **Allosteric site:** Binds with organic modifier (activator or inhibitor). ## Theories of Enzyme Action 1. **Induced fit theory:** - When the substrate approaches the enzyme, the active site becomes similar to the substrates so the substrate fits into the enzyme forming enzyme- substrate complex that gives enzyme + product. 2. **Thermodynamic Changes:** - For any substrate to give product, it should be placed at a high energy called energy of activation, enzymes decrease the energy of activation. - Enzyme doesn't affect the equilibrium constant → ΔG is the same. - ΔG = Energy of the products -- Energy of the substrate. ## Types of Reactions - **According to free energy change ΔG, there are 3 types of reactions:** - **Exothermic reactions (Exergonic reactions):** - ΔG is Negative - Accompanied by release of free energy - Irreversible Example: ATP -> ADP + Pi - **Endothermic reactions (Endergonic reactions):** - ΔG is Positive - Accompanied by gain of free energy - Endergonic reaction should be coupled with exergonic reaction - ATP is the source of energy to most endergonic reactions Example: A + B + ATP -> C + ADP + Pi - **Isothermic reactions:** - ΔG equal zero - Not accompanied with change of free energy - Freely reversible Example: A + B -> C+ D ## Factors Affecting the rate of Enzyme Catalyzed Reaction - Only 1 factor is changed at a time and the remaining factors should remain constant. - The velocity should be measured at the beginning of the reaction (initial velocity or Vi). 1. **Concentration of the Substrate [S]:** - Velocity of reaction: Number of substrate molecules converted to product per unit time. - ↑ [S] → ↑ Velocity of reaction (directly proportional). - **When Vmax (maximal velocity) is reached further in [S]→ No ↑ in velocity of reaction.** - **Km (Michaelis constant):** Substrate concentration that produce 1/2 Vmax. - **At Vmax, all enzymes are saturated with substrates so further ↑ in [S] → No ↑ in reaction velocity.** - **Before Km:** The reaction is responsive to ↑ [S] → Linear curve. - **Beyond Km:** The reaction is less responsive to ↑ [S] → Non-linear curve. **Michaelis-Menten equation:** Describes the behavior of many enzymes when [S] is changed. **Vi = Vmax [S] / Km + [S]** 2. **Significance of Km:** - Index for the affinity of the enzyme to substrate (The lower the Km the higher the affinity). - Isoenzymes have different Km. - If there is an enzyme can bind to more than 1 substrate, it will have different Km for each Substrate. - If there is an inhibitor that binding of the substrate to the enzyme, it will ↓ the affinity ↑ Km. 3. **Concentration of the enzyme [E]:** - ↑ [E] → ↑ Velocity of the reaction till a certain point (Vmax). - Beyond the Vmax→ Further ↑ in [E] → No ↑ in Vi. - The substrate concentration is the limiting factor. 4. **Concentration of the cofactors [C]:** - ↑ [C] → ↑ Velocity of the reaction till a certain point (Vmax). - Beyond the Vmax→ Further ↑ in [C] → No ↑ in Vi. - The enzyme concentration is the limiting factor. 5. **Concentration of the products [p]:** - ↑ Concentration of the products → ↓ velocity of reaction. - ↓ Concentration of the products → ↑ velocity of reaction. 6. **Temperature:** - The optimum temperature for most enzymes is 37°C. - Below this temperature → ↓ Velocity due to collision reaction rate Between the enzyme and substrate i.e., at 0° C → Reaction stops. - At 70° C → Reaction stops due to denaturation. - **NB:** Optimum temperature for plant enzymes is 50° 7. **pH:** - Each enzyme has its optimum pH at which the reaction reaches maximum velocity. - Below or beyond this optimum pH the reaction ↓. - The optimum pH for most enzymes is between 5 and 9 But the optimum pH for pepsin is 2. - At pH 2 units above or below the optimum pH reaction stops. ## Inhibitors **Def:** Any substance that can ↓ the activity of enzyme catalyzed reaction. **Types of enzyme inhibitors:** 1. **Competitive inhibitors** 2. **Allosteric inhibitors** 3. **Other inhibitors** ### 1. Competitive Inhibitors - The inhibitor is similar (structural analogue) to the substrate. - The inhibitor competes with the substrate for binding with the active site of the enzyme → ↓ the rate of chemical reaction. - The inhibition depends on the concentration of the inhibitor and the substrate → ↑ the substrate concentration will remove the inhibition (Reversible inhibitor). **Effect on Km and Vmax:** - ↑ Km - No effect on Vmax **Examples of competitive inhibitors:** 1. **Sulfonamide or Sulfanilamide:** Bacteriostatic (Used in treatment of bacterial infection) - As it is similar to PABA (Para Amino Benzoic Acid) so it competes with PABA for the enzyme that converts PABA to folic acid →→ Bacterial multiplication. 2. **Allopurinol:** Used in treatment of gout (Uric acid formation) - As it is similar to hypoxanthine, so it competes with hypoxanthine for the enzyme xanthine oxidase → ↓ Uric acid formation. 3. **Dicumarol and warfarin:** Anticoagulant - As it is similar to vit K so competes with vit K for the enzyme vit K reductase → active form of vit. K→ ↓ Activation of blood clotting factors ↓ Coagulation. 4. **Statins:** Competitive inhibitor to HMG CoA reductase (key enzyme of cholesterol synthesis). - As it is similar (structural analogue) to HMG CoA, competes with HMG CoA for binding with HMG CoA reductase inhibiting the reaction → ↓ plasma cholesterol. ### 2. Allosteric Inhibitors - Enzymes contain certain site known as allosteric site. - Binding of organic modifier to the allosteric site may produce conformational changes in the active site → decreasing the affinity of the enzyme to the substrate. **Effect on Km and Vmax:** - Km: ↑ (as it affinity of the enzyme to the substrate). - Vmax: ↓ (as it ↓ catalytic activity). **Example:** ATP is allosteric inhibitor for PFK1 (phosphofructokinase 1). **Feedback Inhibition:** - Type of allosteric inhibition. - The end-product of a series of reactions inhibits an enzyme early in the pathway. - Occurs at the earliest irreversible step unique to that particular pathway. **In the case of branched biosynthetic pathways, multiple feedback loops are more common of regulation of enzymes of these pathways.** - **Cumulative feedback inhibition:** When more than 1 product inhibits the same enzyme. - **Short loop feedback inhibition:** The product inhibits the previous enzyme. - **Long loop feedback inhibition:** The product inhibits an enzyme early in the pathway. **Competitive inhibitors:** | | Competitive inhibitors | Allosteric inhibitors | |---|---|---| | Mechanism of action | Inhibitor is similar to substrate and binds to same substrate binding site | Inhibitor is not similar to substrate and binds to allosteric site | | Effect on Km | Increase | Increase | | Effect on Vmax | Same | Decrease | | Examples | Allopurinol is inhibitor for xanthine oxidase uric acid, Statins inhibitor for HMGCoA reductase cholesterol | ATP inhibitor for PFK1 | ### 3. Other Inhibitors **A. Inhibitors that exert their effects on cofactors or prosthetic group:** 1. **Fluoride:** - Inhibits enzymes that require Ca+2 and Mg+2. - Binds and chelates Mg+2 so inhibits enolase leads to inhibition of glycolysis. 2. **Cyanide and carbon monoxide:** - Bind Fe+2 so inhibit cytochrome oxidase. **B. Inhibitors that exert their effects on apoprotein part of the enzyme:** - Nonspecific inhibitors. - Either anti enzymes, factors that denature protein or that block chemical groups in the Active site (enzyme poison). 1. **Antienzymes:** - Enzymes that inhibits another enzyme. - As anti-thrombin III that inhibits thrombin (inhibits coagulation) and activated by heparin. 2. **Inhibitors that denature proteins:** - As strong acids, alkalis, alcohols, and salts of heavy metals. 3. **Chemical group inhibitors (enzyme poison):** - **SH (sulfhydryl) group inhibitors:** - SH group is commonly found at the active site of many enzymes. - SH group inhibitors include: - **Oxidizing agents:** Oxidizes the sulfhydryl group (SH) into disulfide (S-S). - **Salts of heavy metals:** As HgCl₂ (mercury chloride) - The +ve charged heavy metal binds to the -ve charged sulfur of SH. - **Alkylating agents as iodoacetate:** alkylates the SH group - **OH (Hydroxyl) group inhibitors** - OH group is commonly found at the active site of many enzymes. - **Examples:** - Aspirin: Produces acetylation of OH of serine of cyclo-oxygenase (COX)→↓PG synthesis → anti-inflammatory and antipyretic action of aspirin. ## Regulation of Enzyme activity - Regulation of enzyme activity is achieved by 3 mechanisms: 1. Changing the amount of the enzyme. 2. Changing the activity of the enzyme. 3. Compartmentation of enzymes. ### 1. Changing the amount of the enzyme: - The amount of the enzyme is controlled by rate of: - **Enzyme synthesis:** - ↑ Synthesis: by ↑ gene expression of the enzyme (induction) by inducer which may be the substrate of the enzyme or hormone. - ↓ Synthesis: by ↓ gene expression of the enzyme (Repression) by repressor which may be the product or hormone. - **Enzyme degradation:** - By controlling the synthesis or the activity of the enzymes responsible for degradation. ### 2. Changing the activity of the enzyme: - **A. Activation of zymogens (Proenzymes):** - Def.: They are enzymes secreted in an inactive form. - As the Active Site is masked by polypeptide chain. - Activated by removal of these polypeptide chain. - The active form of the enzyme can then activate its zymogen (autocatalysis or autoactivation). - Examples: - Digestive enzymes and enzymes of blood clotting factors (activated by proteases). - Example: Pepsinogen - HCL.→ Pepsin - autocatalysis - **Most of blood clotting factors are formed as zymogens and activated by: specific proteases** - **B. Allosteric modifiers (Inhibitors and activators):** - **Allosteric inhibitor:** Explained before. - **Allosteric activator:** Binds to the allosteric site→ Becomes more suitable to bind with the substrate→ ↑ affinity. - **Effect on Km and Vmax:** - KM:↓ - Vmax: ↑ - **Examples:** AMP is allosteric activator of PFK1. - **C. Covalent modification:** - Activation and inhibition by phosphorylation and dephosphorylation. - Many enzymes are activated by phosphorylation and inhibited by dephosphorylation and vice versa. - The enzyme is present in 2 interconvertible forms (phosphorylated and dephosphorylated). - Phosphorylation occurs by kinase and dephosphorylation by phosphatase. - Phosphate is attached to OH of serine, threonine, or tyrosine. ### 3. Compartmentation of enzymes: - Cells and subcellular organelles are separated by membranes. - The transport across these membranes is under control and regulation. - Localization of metabolic reactions (enzymes) in cytosol or subcellular organelles facilitate regulation of these pathways (FA synthesis in the cytosol while FA oxidation in the mitochondria). ## Classification of Enzymes - IUBMB (International Union of Biochemistry and Molecular Biology) developed a system of Nomenclature for enzymes. - Enzymes are classified into 6 classes: - **Hydrolases:** Break by addition of water. - **Oxidoreductases:** For oxidation reduction reactions. - **Transferases:** Transfer chemical group from compound to another compound. - **Lyases:** Break down without addition of water. - **Isomerases:** Convert isomer to another isomer. - **Ligases:** Bind 2 compounds together.