Biochem Lec_ PPT 4 and 5.pdf

ENZYME TERMINOLOGIES Inhibition - process that makes an active enzyme less active of inactive Enzymes-biological catalysts - Is a compound, usually a protein, that acts as catalysts for biochemical r...

ENZYME TERMINOLOGIES Inhibition - process that makes an active enzyme less active of inactive Enzymes-biological catalysts - Is a compound, usually a protein, that acts as catalysts for biochemical reactions CHARACTERISTICS OF ENZYMES - “Catalyst” - something that makes a 1. Predominantly protein in nature (except chemical reaction happen more quickly ribozymes which are made of ribonucleic without itself being changed. Can be used acid) 9 over and over in a reaction 2. Increase the rate of a reaction by 10 to 20 10 Substrate - compound/s whose reaction an 3. Commonly end with -ase (example: urease, enzyme catalyzes sucrase, lipase) - The substance upon which the enzyme “acts” - Enzyme act on your substrate to convert substrate into a product On the left of the reaction: Substrate Conjugated enzyme - an enzyme that has a non On the right: Products protein part in addition to a protein part Above or beside the arrow: Enzyme Below the arrow: Cofactors or Coenzymes Coenzyme - non-protein organic molecule, frequently a B vitamin, that acts as a cofactor - A small organic molecule that serves as a ENZYMES MECHANISM OF ACTIVITY cofactor in a conjugated enzyme that enhances the catalytic function of an enzyme Cofactor - non-protein part of an enzyme that is necessary for catalytic function (eg Zn and Mg) - Minerals Apoenzyme -protein part of an enzyme Prosthetic Group - the coenzyme or cofactor that 1st Step: Enzyme + Substrate = Reversible is tightly bound to the apoenzyme Enzyme-Substrate Complex 2nd Step: Enzyme-Substrate Complex -> Holoenzyme - apoenzyme + prosthetic group Formation of the product and regineration Active site - specific portion of the enzyme to which a substrate binds during reaction SPECIFICITY OF ENZYMES Bond Specificity Activation - any process that initiates or increases Group Specificity the activity of an enzyme - The enzymes will act only on molecules that have a specific Allosteric site - portion on the enzyme surface functional group where inhibitors/activators bind to regulate catalytic Substrate Specificity reactions Optical Specificity - Any other site where other molecules can - L and D amino acids bind to the enzyme aside from the active Geometrical Specificity site - Shape Co-factor Specificity Inhibitor - compound/s that slows down the rate of the reaction - A substance that slows or stops the normal catalytic function of an enzyme by binding to it MECHANISMS OF CATALYSIS 1. Lock-and-Key Model Collision Theory: For a reaction to proceed, there - The active site in the enzyme has a should be an effective collision between the fixed, rigid geometrical conformation. molecules of the reactants. If you mix two reactants Only substrates with a together, during collision, they produce a collision complementary geometry can be energy of these two reactants that should at least accommodate at such a site, much be equal to the activation energy. as a lock accepts only a certain key Transition State Theory: You have 2 reactants. As 2. Induced-Fit Model reactant 1 approaches reactant 2. Nagkakaroon ng - Many enzymes have flexibility in attraction and repulsion. This changes the energy their shapes. They are not rigid and and even the shape/structure (transition state). As static; there is constant change in the two reactants become close together, the their shape. energy requirement is reached. Effective reaction - Allows for small changes in the and formation of products is achieved. Transition shape or geometry of the active site state is equivalent to the energy required for a of an enzyme to accommodate a reaction to happen (activation energy) substrate Catalytic Reaction: From a high energy activation, it lowers the activation energy. This lowering of the ENZYME ENERGETICS activation energy increases the rate of the reaction. CLASSIFICATION OF ENZYMES 1. Oxidoreductase: catalyzes an oxidation - reduction reactions - Example is lactate dehydrogenase that removes hydrogen atoms from a molecule - Requires an electron donor or electron acceptor - Presence of NAFH and FADH2 - “Dehydorgenase” - the electrons are removed or added in the form of hydride 2. Transferase: catalyzes the transfer of a functional group from one molecule to another - Amino group (NH3) is transferred - 2 substrates and 2 products 3. Hydrolase: breaking of bonds in the presence of water - Presence of H2O (may be) then 2 products - Breakage of ester bonds  4. Lyase: addition or removal of double/ triple bonds : catalyzes the addition of a group to a double bond or the removal of a group to form a double bond in a manner that does not involve hydrolysis or oxidation - Water is also present to provide hydroxyl group (OH) 5. Isomerase: isomerization reaction : catalyzes the isomerization (rearrangement of atoms) of a substrate in a reaction, converting it into a molecule isomeric with itself 6. Ligase: formation of new bonds : catalyzes the bonding together of two molecules into one with the participation of ATP - 2 or more substrates form 1 product ENZYME ACTIVITY - Measure of how fast the enzyme-catalyzed reaction happen - How fast the substrate are utilized - How fast the products are formed Reactants: negative sign - Since they are being used up. Change is negative. Substrate concentration decreases Products: positive - Since nadadagdagan FACTORS AFFECTING ENZYME ACTIVITY 1. Effect of Enzyme Concentration Proportional increase in enzyme concentration and rate of reaction  - More enzymes mean more molecules to convert substrate to products The greater the enzyme concentration, the greater the reaction rate 2. Effect of Substrate Concentration At low substrate concentration, directly proportional - first order kinetics At high substrate concentration, the increase in rate of the reaction plateau because of saturation - zero order kinetics - Kahit anong increase in substrate concentration, rate of the reaction remains constant/fixed - Enzyme activity increases up to a certain substrate concentration and therefore remains constant - As substrate concentration increases, the point is eventually reached where enzyme capabilities are used to their maximum extent The effect of substrate concentration to enzyme activity is the basis of the catalytic parameters Vmax and KM - Vmax - maximum velocity; highest rate of the reaction at desaturation point - KM - substrate concentration at half Vmax COMPUTATIONS: Affinity - strength of binding of the substrate to the enzyme Mas mabilis magbind ang substrate to enzyme if KM is lower  Reciprocal of Michaelis-Menten Equation The higher the Kcat, the higher the turnover number. Mas mabilis magconvert ng product 3. Effect of Temperature As the temperature of an enzymatically catalyzed reaction increases, so does the rate (velocity) of the reaction However, when the temperature increases beyond a certain point, the increased energy begins to cause disruptions in the tertiary structure of enzymes; denaturation is occurring Optimum temperature is the temperature at which an enzyme exhibits maximum capacity. When the enzyme works best 4. Effect of pH Changes the ionization state Change in pH alters the charge of amino acid residues found in active site. Optimal pH: pH at which an enzyme exhibits maximum activity (achieves Vmax) Vmax: maximum reaction rate Extreme pH (too acidic/basic): denatured enzyme irreversibly; loss of catalytic activity. 5. Effect of Inhibitors Types of Inhibitors: Competitive Inhibitor - Directly binds to the active site - Competes against the substrate in binding to the active site  - Reversible inhibitor because if you increase the substrate concentration, the chances of the substrate being able to bind to the active site will also increase. Hence, reversing the inhibition caused by - Can be overcome by increasing the substrate concentration Non-Competitive Inhibitors - Binds to allosteric sites - pockets on the surface of the enzyme where inhibitors or regulators bind. If an inhibitor binds to the active site, it will change its structure. If you change the structure, then the substrate can no longer bind therefore there will be no more catalysis - Alters the enzyme conformation SUMMARY Enzymes are biological catalyst which are predominantly protein in nature Enzymes speeds up the reaction by lowering the activation energy Catalysis happens via a two-step process. The first is enzyme-substrate complex formation and the second is conversion of substrate to product. Enzymes are classified according to the type of reaction they catalyzed. The six classifications are oxidoreductase, transferase, hydrolase, ligase, lyase and isomerase Enzyme activity is measured in terms of KM. Vmer and Keat Enzyme activity is affected by enzyme concentration, substrate concentration, temperature, pH and presence of inhibitors. Competitive inhibitors compete against the substrate in binding to the active site. Non-competitive inhibitors bind to allosteric region affecting the enzyme conformation and binding of the substrate to the active site ENZYMES INHIBITION AND UNCOMPETITIVE INHIBITORS REGULATION BInds directly to ES complex, but not to free E; Distorts the active site, affecting catalytic REVIEW function but not its S binding Enzymes are biological catalysts, meaning Adding S does not reverse the effect they speed up a chemical reaction’ Significant only for multi-substrate enzymes Enzymes increase the rate of reaction by Both KM and Vmax are lowered lowering the activation energy Does not compete on active site but does Enzymes have active site (where the not allow it to produce a product by binding substrate binds) and allosteric site (also to the enzyme substrate called regulatory site; where the regulator molecule binds) ENZYME REGULATION Mechanisms of Catalysis: Lock and Key, 1. Feedback Inhibition and Induced Fit 2. Allosteric Regulation 3. Covalent Modification FACTORS AFFECTING ENZYME ACTIVITY 4. Proenzyme/ Zymogen Enzyme concentration 5. Isoenzyme Forms Substrate concentration 6. Genetic Expression Induction pH Temperature Feedback Inhibition Presence of Inhibitors Applicable in multi-step (intermediary - Competitive Inhibitors metabolic) pathways - Non-competitive inhibitors An end product inhibits key enzymes which - Mixed competitive forms the intermediates - Uncompetitive The final product of a series of pathways serve as inhibitors of the enzymes which are part of the process of its synthesis COMPETITIVE INHIBITORS Binds directly on the active site BInds only to free E (enzyme); reducing [E] available for substrate binding Effect could be reverse by increasing [S] (Substrate Concentration) because by increasing this, you will reach the Vmax and the highest rate is achieved. KM is increased since need niya makipagcompete KM is raised; Vmax same NON-COMPETITIVE INHIBITORS Interacts either to E or ES (Enzyme-Substrate Complex) Binds to allosteric sites Cannot be overcome by increase in [S] KM same; Vmax is lowered Aspartate transcarbamoylase (ATCase), an Does not compete on the active site but it enzyme which initiates the reaction for the changes the conformation of enzyme by synthesis of cytidine triphosphate (CTP), is binding on the allosteric site inhibited by CTP - CTP is one of the precursors for MIXED NON-COMPETITIVE INHIBITORS DNA synthesis. Pag marami ng CTP Similar to noncompetitive ang naproduce from this reaction, Binding of I (Inhibitor) affects binding of S CTP itself serve as an inhibitor of (Substrate) that enzyme, therefore it will now KM and Vmax are lowered prevent the production of CTP.  active site of that subunit. However, if it is in R, the substrate can easily bind on the active site of the enzyme - In some cases, some portions will be in the R conformation, some will be on T. It's one way to regulate your enzymes. Inhibitors are used in regulating enzymes. - If you want to stop the activity, it needs an allosteric inhibitor that will favor the taut conformation - If you want to activate an enzyme, you will need an inhibitor that will favor the relaxed conformation Allosteric Regulation Some multi-subunit enzyme have catalytic sites and regulatory sites Allosteric regulators bind to regulatory sites to regulate catalytic activity - Example is the ATCase that contains Concerted Model 3 catalytic trimer (contain active site) In the absence of a substrate, an and 3 pairs of regulatory dimer equilibrium between T and R conformation (involved in the regulation). The exists catalytic trimer helps in the synthesis Presence of substrate affects the of CTP. Regulatory dimer serves as equilibrium towards R conformation the region for the binding of either of CTP or ATP to regulate the activity Effect of Activators and Inhibitors in T and R The role activators and inhibitors in the Conformation kinetic of the enzyme Allosteric inhibitors bind to favor T - ATP is an activator of ATCase conformation - CTP is an inhibitor of ATCase Activator bind to favor R conformation Sequential Model Sequential activation of the subunits upon Taut (T) and Relaxed (R) Conformation binding of a substrate to one of the catalytic Conformational transitions affect substrate sites of a multi-domain enzyme binding T conformation, low affinity Covalent Modification R conformation, high affinity Introduction of a group to the enzyme either - When a subunit of an enzyme is at activate or deactivate the enzyme the T conformation, the substrate will - Phosphorylation have a difficulty in binding to the - Methylation  Genetic Induction TRANSITION STATE ANALOG Have the same structure as the substrate in the ES complex Have lower energy requirement compared to substrate Preferred by enzyme compared to substrate TRANSITION STATE ANALOGS AS ANTIVIRALS 2-deoxy-2,3-dehydro-N - acetylneuraminic acid (DANA) Substrate for neuraminidase essential for viral glycoprotein/glycosaminoglycan synthesis Zymogen Zymogen is an inactive precursor of enzymes Pepsinogen Trypsinogen Chymotrypsinogen Isoenzyme Forms Enzymes in different cell types may have different isoforms Glucose transporters (GLUT) - GLUT1 (erythrocyte) - GLUT2 (liver) - GLUT3 (brain) - GLUT4 (muscle) VIRAL PROLIFERATION CAN BE STOPPED BY INHIBITING STEPS IN IN ITS LIFE CYCLE