MBC WK1.5.pdf

COMMONWEALTH OF AUSTRALIA Copyright Regulations 1969 WARNING This material has been reproduced and communicated to you by or on behalf of La Trobe University pursuant to Part VB of the Copyright Act 1968 (the Act)....

COMMONWEALTH OF AUSTRALIA Copyright Regulations 1969 WARNING This material has been reproduced and communicated to you by or on behalf of La Trobe University pursuant to Part VB of the Copyright Act 1968 (the Act). The material in this communication may be subject to copyright under the Act. Any further reproduction or communication of this material by you may be the subject of copyright protection under the Act. Do not remove this notice. Slide 1  Module 1 – Enzymes Dr Lakshmi Wijeyewickrema Department of Biochemistry and Genetics Slide 2 Week 2 Lecture 4 Slide 3 Intended Learning Outcomes (ILOs) After this lesson students will be able to: Understand that enzymes can be inhibited by specific molecules Understand the different forms of reversible inhibitors Understand an irreversible enzyme inhibitor is a molecule that inactivates enzymes by forming a strong covalent bond to an amino acid side-chain group at the enzyme’s active site. Slide 4  Enzyme Inhibition ENZYME INHIBITOR A substance that slows down or stops the normal catalytic function of an enzyme by binding to the enzyme Three types of inhibition: Competitive inhibition Uncompetitive inhibition Irreversible inhibition  Enzyme Inhibition Competitive Inhibition A competitive inhibitor resembles the substrate Inhibitor competes with the substrate for binding to the active site of the enzyme If an inhibitor is bound to the active site: Prevents the substrate molecules to access the active site Decreasing / stopping enzyme activity. The binding of the competitive inhibitor to the active site is a reversible process. Adding much more substrate can outcompete the competitive inhibitor. Many drugs are competitive inhibitors: Anti-histamines inhibit histidine decarboxylase, which converts histidine to histamine Department of Biochemistry and Genetics Slide 6  Enzyme Inhibition Uncompetitive Inhibition An uncompetitive inhibitor decreases enzyme activity by binding to the enzyme substrate (ES) complex preventing the conversion of substrate to product. Adding substrate increases inhibitor effect Example: A few pesticides are uncompetitive inhibitors, the best-known example being the herbicide N-phosphonomethylglycine, commonly known as glyphosate or Roundup, an uncompetitive inhibitor of 3- phosphoshikimate 1- carboxyvinyltransferase. Uncompetitive inhibitor Department of Biochemistry and Genetics Slide 7  Enzyme Inhibition Irreversible Inhibition An irreversible inhibitor inactivates an enzyme by binding to its active site by a strong covalent bond – Permanently deactivates the enzyme – Irreversible inhibitors do not resemble substrates The addition of excess substrate doesn’t reverse this process – Cannot be reversed Chemical warfare (nerve gases) Organophosphate insecticides Department of Biochemistry and Genetics Slide 8 Chymotrypsin is a protease. It hydrolyses peptide bonds in proteins and peptides. The enzyme acts in the digestive tract to yield small peptides and amino acids. Two amino acids in the active site are particularly important for catalysis, Ser195 and His57  Irreversible inhibition of a serine protease by diisopropylfluorophosphate (DIFP). DIFP reacts with the active site serine (Ser195) to form DIFP-chymotrypsin.  Summary Enzyme inhibitors are substances that bind to an enzyme and stop or slow its normal catalytic activity. A Competitive inhibitor is molecule closely resembling the substrate. Binds to the active site and temporarily prevents substrates from occupying it, thus blocking the reaction. An Uncompetitive inhibitor binds to a site on an enzyme that is not the active site. The normal substrate still occupies the active site, but the enzyme cannot catalyse the reaction due to the presence of the inhibitor. An Irreversible inhibitor forms a covalent bond to a part of the active site, permanently preventing substrates from occupying it. Slide 11 Resources Lehninger Principles of Biochemistry Seventh Edition (2017), W. H. Freeman and Company Chapter 6.3 Slide 12 Week 2 Lecture 5 Slide 13 Intended Learning Outcomes (ILOs) After this lesson students will be able to: Basics of enzyme kinetics graphs What does the Km mean? What is the importance of a kcat value? Revision of enzyme inhibition Example of an enzyme kinetic problem Slide 14 S, P, AND E (SUBSTRATE, PRODUCT, ENZYME) Enzyme (E) converts substrate(s) (S) to product(s) (P) and accelerates the rate. slow No conversion S No (E) P A S-to-Pase B (E) Quick, specific S S-to-Pase P (E) ACTIVE SITE The active site is the special place, cavity, crevice, chasm, cleft, or hole that binds and then transforms the substrate to the product. The kinetic behaviour of enzymes is a direct consequence of the protein’s having a limited number (often 1) of specific active sites. ASSAY An assay is the act of measuring how fast a given (or unknown) amount of enzyme will convert substrate to product—the act of measuring a velocity. VELOCITY Velocity—rate, v, activity, —is how fast an enzyme converts substrate to product, the amount of substrate con- sumed, or product formed per unit time. Units are micromoles per minute (µmol/min).  VELOCITY The VELOCITY of product formation (or substrate disappearance) is defined as the change in product concentration per unit time. It is the slope of a plot of product concentration against time. The velocity of product formation is the same as the velocity of substrate disappearance (except that substrate goes away, whereas product is formed). INITIAL VELOCITY This is the measurement of the rate under conditions under which there is no significant change in the concentration of substrate. The velocity is not necessarily the same at all times after you start the reaction. The depletion of substrate, inhibition by the product, or instability of the enzyme can cause the velocity to change with time. The initial velocity is measured early, before the velocity changes. Initial velocity measurements also let you assume that the amount of substrate has not changed and is equal to the amount of substrate that was added.  The initial velocity of an enzyme reaction is dependent on substrate concentration Substrate concentration affects the velocity of an enzyme catalysed reaction. Almost all enzyme-catalyzed reactions show saturation behavior. At a high enough substrate concentration, the reaction just won’t go any faster than Vmax. The substrate concentration required to produce a velocity that is one-half of Vmax is called the Km. Michaelis-Menten equation and constants Above is the Michaelis-Menten equation Km is defined as the substrate concentration at half Vmax (units = M [concentration]) Vmax is the theoretical maximum velocity of an enzyme reaction at a given enzyme concentration (units = M-1s-1) The Km indicates the substrate concentration at which half-maximal velocity will be reached. In order for an enzyme substrate reaction to be physiologically relevant, it is clear that the enzyme must be functioning at a reasonable rate of reaction. The Km of the enzyme for the substrate should therefore be close to or lower than the physiological concentration range of the substrate. Substrates that bind tightly have small Km values. Substrates that bind weakly have large Km values. Simplistically, Km is a measure of the affinity of the enzyme for its substrate. The higher the Km, the lower the affinity (enzyme-substrate binding is less strong).  Vmax This is the velocity approached at a saturating concentration of substrate. Vmax has the same units as v. kcat = Vmax/[E]. kcat is equal to the slowest step in the reaction: kcat is often assumed to relate to the conversion of ES to E + P, but in fact it relates to whichever step in the reaction is slowest. It is the rate at which the enzyme turns over substrate to produce product and therefore a measure of the enzyme’s efficiency in this process. The Michaelis-Menten plot can be rearranged to allow a linear plot. This allows easier determination of the kinetic values, Km and Vmax, using the LINEWEAVER-BURK PLOT for instance.  Enzyme inhibition Enzymes can be prevented from carrying out their function by INHIBITORS Irreversible, often a covalent modification of the enzyme. Reversible, has ability to associate and dissociate from the enzyme Competitive Inhibition Inhibitor competes for the same site as the substrate. Vmax is unchanged Km is increased (binding of substrate is decreased).  Shape and structure of inhibitor is very similar to substrate Competitive Inhibition Inhibitor mimics substrate and fits into the active site Physically blocks substrate’s access into the active site Vmax is unchanged because increasing the [S] can overcome the inhibitor. Km increases ( affinity between enzyme and substrate decreases) because the inhibitor is taking some of the enzyme ‘out of action’ Slide 30 Uncompetitive Inhibition Inhibitor only binds to ES complex; immediately means I binds to a different site from S Km and Vmax are changed Uncompetitive The inhibitor binds to a site other than the active site, but only if the ES Inhibition complex is already formed. Distorts the active site and thus inactivates the enzyme. Prevents release of the substrate from the binding site. Vmax reduced because enzyme is prevented from forming products Km reduced (affinity between enzyme and substrate increases) because the inhibitor makes the reaction favour the ES complex Slide 32  Summary The Michaelis–Menten equation relates the initial velocity of a reaction to the maximal reaction velocity and the Michaelis constant for a particular enzyme and substrate. A Lineweaver–Burk plot can be used to present kinetic data and to calculate values for Km and Vmax. Km is the substrate concentration at which the reaction velocity is half-maximal. The value of kcat/Km indicates an enzyme’s catalytic efficiency. Kinetic data can be plotted in double-reciprocal form to determine Km and Vmax. Reversible inhibitors reduce an enzyme’s activity by binding to the substrate-binding site (competitive inhibition), or to the enzyme–substrate complex (uncompetitive inhibition). Slide 33 Resources Lehninger Principles of Biochemistry Seventh Edition (2017), W. H. Freeman and Company Chapter 6.3 Slide 34 Week 2 Lecture 6 Slide 35 Enzyme Regulation: Allosteric enzymes Enzymes whose activity can be changed by molecules other than a substrate. Allosteric enzymes have a quaternary structure – Are composed of 2 or more protein chains – Possess 2 or more binding sites 2 types of binding sites: One binding site for the substrate - Active site Second binding site for a regulator molecule - Regulatory site Active & regulatory binding sites are distinct from each other in shape & location An allosteric enzyme is an enzyme with two or more protein chains (quaternary structure) and two kinds of binding sites (substrate and regulator) and can exist in two forms. Mechanisms of Allosteric Inhibition and Activation. Allosteric enzymes do not show a Michaelis- Menten relationship between [S] and V0 Most enzymes show Michaelis- Menten Allosteric enzymes do not follow kinetics, in which the plot of initial Michaelis-Menten kinetics and show a reaction velocity (vo) against substrate sigmoidal curve. concentration ([S]), is hyperbolic. Slide 38 Allosteric enzymes do not show a Michaelis- Menten relationship between [S] and V0 Allosteric enzymes do not follow The effects of a positive modulator (+) Michaelis-Menten kinetics and show a and a negative modulator (-) on an sigmoidal curve. allosteric enzyme in which K0.5 is altered without a change in Vmax. The central curve shows the substrate-activity relationship without a modulator. Slide 39 Allosteric enzymes larger and more complex, have two or more subunits. do not follow Michaelis-Menten Kinetics. Have two conformations, one active and one inactive Binding of activator stabilises the active site and the active conformation – enzyme works. Binding of inhibitor stabilises the inactive site form – enzyme does not work. Are regulated by allosteric activators or inhibitors. Can be up-regulated by allosteric activators at constant [S]. Can be down regulated by allosteric inhibition at constant [S]. Enables them to be ‘fine-tune’ and amplify their catalytic ability when required. An essential characteristic for their roles as regulatory enzymes that govern the overall rate of the metabolic pathways to ensure promptness in the response to the cells’ needs. Slide 40 Resources Lehninger Principles of Biochemistry Seventh Edition (2017), W. H. Freeman and Company Chapter 6.1 Slide 41