SL12001 Biochemistry Lecture 9 PDF

SL12001 BIOCHEMISTRY Dr Scott Lovell [email protected] 4 South 1.30 Topic 3: Enzymes Lecture 7: Basic concepts – catalysis, transition states and binding energy Please provide Lecture 8: Enzyme...

SL12001 BIOCHEMISTRY Dr Scott Lovell [email protected] 4 South 1.30 Topic 3: Enzymes Lecture 7: Basic concepts – catalysis, transition states and binding energy Please provide Lecture 8: Enzyme classes and enzyme kinetics feedback! Lecture 9: Enzyme inhibition and regulation Michaelis-Menten Kinetics Why is this important? It accounts for the kinetic data that we described previously When [S] > KM the equation simplifies to: I.e., the initial rate is independent of [S] When [S] = KM the equation simplifies to: The KM is the substrate concentration at which the reaction rate is half the maximal rate Measuring KM and Vmax (1/2) Vmax is only approached but never attained. How can we experimentally determine KM and Vmax? We measure the initial rate of catalysis, V0, at several [S] and then use curve-fitting programs on a computer Measuring KM and Vmax (2/2) An older method is to manipulate the Michaelis-Menten equation to afford a straight-line plot that yields the values for Vmax and KM. Taking the reciprocal of both sides of the equation gives: A plot of 1/V0 vs 1/[S], called a Lineweaver- Burk plot, provides a straight-line with a y- intercept of 1/Vmax and a slope of KM/Vmax. The intercept on the x-axis is -1/KM. MentiMeter Quiz 1 Lecture 9 Learning Objectives 1) Recap Michaelis-Menten Kinetics 2) Introduce the various classes of reversible enzyme inhibitor 3) Recognize how irreversible enzyme inhibitors differ from reversible enzyme inhibitors 4) Assess how enzymes are regulated in cells and tissues Enzyme Inhibition - Molecules that interfere with enzyme catalysis, slowing or halting enzymatic transformations - Enzyme inhibitors are important pharmaceutical agents and have been used to treat a variety of disease such as cancer, HIV and malaria. Specific Non-Specific e.g., by Acid Reversible Irreversible Ethanol Heavy metal Competitive Non-competitive Uncompetitive Reversible Enzyme Inhibition Competitive Inhibition (1/2) - A competitive inhibitor competes with the substrate for the active site of the enzyme KI = [E] [I] / [EI] - KI, the inhibitor constant, is an indication of how potent an inhibitor is - KI = the [I] required to produce half maximum inhibition - The bound inhibitor does not inactivate the enzyme. Competitive Inhibition (2/2) Michaelis-Menten Equation for Competitive Inhibition KI = [E] [I] / [EI] KM Vmax = unchanged - ⍺KM = the observed KM in the presence of inhibitor, - referred to as the apparent KM - When [S] >> [I] the reaction will still be at Vmax - In the presence of inhibitor higher [S] are required to reach the Vmax - KM increases in the presence of inhibitor Example of a competitive inhibitor - Ethanol is used to treat patients with methanol poisoning - Methanol CH3OH is found in antifreeze and when ingested is converted to formaldehyde (CH 2O) by alcohol dehydrogenase - Formaldehyde is extremely toxic and causes blindness Methanol Ethanol Non-competitive Inhibition (1/2) - A non-competitive inhibitor binds to the free enzyme and the enzyme-substrate complex but at a different site to the substrate KI = [E] [I] / [EI] KI’ = [ES] [I] / [ESI] - The bound inhibitor does not inactivate the enzyme. Non-competitive Inhibition (2/2) KI = [E] [I] / [EI] Michaelis-Menten Equation for Competitive Inhibition KI’ = [ES] [I] / [ESI] KM = unchanged Vmax - The substrate can still bind to Enzyme-Inhibitor complex, but the ESI complex does not progress to product - The inhibitor lowers the concentration of functional enzyme, so the Vmax is decreased (Vmax = k2[E]T). - The substrate affinity for the enzyme is unchanged so the KM remains the same - Non-competitive inhibition cannot be overcome by increasing [S] Link to schematic Uncompetitive Inhibition (1/2) - An uncompetitive inhibitor binds at a site distinct from the substrate active site and binds only to the ES complex S KI’ = [ES] [I] / [ESI] S - The bound inhibitor does not inactivate the enzyme. Uncompetitive Inhibition (2/2) Michaelis-Menten Equation for Uncompetitive Inhibition KM KI’ = [ES] [I] / [ESI] Vmax - An uncompetitive inhibitor lowers the maximum rate of catalysis, Vmax - An uncompetitive inhibitor also lowers the KM - The inhibitor is ‘removing’ some fraction of enzyme from the reaction - Vmax is decreased because its value depends on [E] (Vmax = k2[E]T) - The KM is lowered because the uncompetitive inhibitor increases the enzyme affinity for the substrate. This can be explained using Le Chatelier’s principle Ref: https://chem.libretexts.org Determining mechanism of inhibition with Lineweaver-Burke Plots - Each class of inhibitor has a different effect on Km and Vmax and therefore produces a unique Lineweaver-Burke plot - We can use Lineweaver-Burke plots to elucidate the mechanism of an inhibitor Competitive inhibitor Step 1 Calculate V0 values for Step 2 V0 vs [S] for each [I] Step 3 plot 1/V0 vs 1/[S] each different [I] No [I] 1[S] 2[S] 3[S] 1[S] - Why? We can determine the mechanism of inhibition and can calculate the KI Summary of Reversible Inhibition Competitive Inhibition Non-competitive Inhibition Uncompetitive Inhibition KM KM KM = unchanged Vmax = unchanged Vmax Vmax Irreversible Enzyme Inhibition Irreversible Enzyme Inhibition (1/3) - Irreversible inhibitors covalently modify a functional group on an enzyme that is essential for activity - Let’s look at an example using the covalent inhibition of a serine protease…. Tetrahedral Intermediate Irreversible Enzyme Inhibition (3/3) - Irreversible inhibitors covalently modify a functional group on an enzyme that is essential for activity - Diisopropylfluorophosphate (DIFP) inhibits all serine proteases and is a highly toxic sarin gas analogue Mechanism-based Irreversible Inhibitors (1/2) - Mechanism-based inhibitors hijack the normal enzyme reaction mechanism and can be selective to a single protease KLK3 – serine protease Mechanism-based Irreversible Inhibitors (1/2) - Mechanism-based inhibitors hijack the normal enzyme reaction mechanism and can be selective to a single protease Transition state analogue Enzyme Regulation Enzyme Regulation - The process by which cells can turn on, turn off, or modulate the activity of various metabolic pathways by regulating the activity of an enzyme. - Enzyme regulation permits the changing needs of the cell to meet its energy and resource demands. (a) If a product is available in excess, enzymes are regulated to divert resources elsewhere (b) If a product is in demand, enzymes can be activated to produce more of the required biomolecule Allosteric Regulation Reversible Covalent Modification Proteolytic Cleavage Feedback Regulation Allosteric Enzyme Regulation - Regulatory molecules, called allosteric modulators or allosteric effectors can bind to an enzyme resulting in activation or deactivation. - Binding is usually away from the active site but results in a change in the shape of the active site. Allosteric Activation – the active site Allosteric Deactivation – the active site becomes available for a substrate becomes unavailable for a substrate Reversible Covalent Modification - Enzyme activity is modulated by covalent modification of amino acid residues in an enzyme molecule - Also known as a post-translational modification (PTM) Proteolytic Cleavage - Enzymes regulated by proteolytic cleavage are produced in an inactive form called a zymogen or pro-enzyme - The enzyme becomes active after removal of a polypeptide segment by proteolytic cleavage - Specific cleavage causes conformational changes that form a fully functional enzyme active site Example of Regulation by Proteolytic Cleavage - Kallikrein-related peptidases (KLKs) are secreted by prostate cancer cells and drive cancer invasion and migration Example of Regulation by Proteolytic Cleavage - Kallikrein-related peptidases (KLKs) are secreted by prostate cancer cells and drive cancer invasion and migration Feedback Regulation - The end-product of an enzymatic pathway inhibits an upstream enzyme to decrease rate of production MentiMeter Quiz 2 Lecture 9 Learning Outcomes 1) Recapped Michaelis-Menten Kinetics 2) Introduced the various classes of reversible enzyme inhibitor 3) Recognized how irreversible enzyme inhibitors differ from reversible enzyme inhibitors 4) Assessed how enzymes are regulated in cells and tissues Please contact me with questions/suggestions Dr Scott Lovell [email protected] 4 South 1.30