UPDATED BMS100_BCH1.09_W23_Kinetics 2_STUDENTS (1).pptx

Enzymes Part 2: Reposted Objectives slides were added to beginning Updates were made to the following original slide #’s: #3: Missing “+” and “H” were added to “NAD+” and “NADH” #24: enzyme is “hexokinase” Enzymes Part 2 Kinetics Dr. Heisel BMS100 Kinetics Objectives Define Km, Vmax Define wha...

Enzymes Part 2: Reposted Objectives slides were added to beginning Updates were made to the following original slide #’s: #3: Missing “+” and “H” were added to “NAD+” and “NADH” #24: enzyme is “hexokinase” Enzymes Part 2 Kinetics Dr. Heisel BMS100 Kinetics Objectives Define Km, Vmax Define what is meant by a first order, pseudo-first order, and zero order reaction, and state whether or not each conforms to Michaelis-Menten kinetics Compare and contrast glucokinase and hexokinase Define Kcat and provide the ratio that determines enzyme efficiency Provide examples of physiologically efficient enzymes and their roles Kinetics Objectives Use Michaelis-Menten kinetics and Lineweaver-Burk plots to: Determine the Km and Vmax of an enzyme Distinguish different types of enzyme inhibition Define and provide examples of reversible, irreversible, and allosteric inhibition Use Km and Vmax to different competitive, uncompetitive and non-competitive inhibition Recognize the graph of an allosteric enzyme in the presence and absence of an inhibitor Prereading Review What is meant by a first order reaction? What is happening in a zero-order reaction? What can we use a Lineweaver-Burk plot Vmax has for? been reached, 0 order ? ? ? Application of Km: Assignment Review NAD+ NADH NAD+ NAD + H+ H+ Ethanol Acetaldehyde H+ Acetate E= aldehyde dehydrogen ase Alcohol consumption Flushing Back to normal https://freesvg.org/1455185701 https://commons.wikimedia.org/wiki/File:Smiley_Face.JPG Application of Km and Vmax: Glucokinase Hexokinase (most tissues) contrasts to glucokinase (liver) in the following ways: Lower Vmax Lower Km It is turned off by high concentrations of glucose-6-P Take a moment to discuss the next 3 slides with your classmates Glucokinase and Hexokinase What same reaction (substrate and product) do both hexokinase and glucokinase catalyze? (convert glucose to G-6P) The product can be used for one of two glucose metabolism pathways: One that breaks down glucose = ? Glycolysis One that stores ? glucose?= ? Glycogenesis Product Glucokinase and Hexokinase Which line in the graph represents: Glucokinase Hexokinase A Hexokinase is blue Glucokinase is red Which intercepts (AD) represent: Vmax (A) ½ Vmax (C) Km (D) Abali, Emine E; Cline, Susan D; Franklin, David S; Viselli, Susan M. Lippincott Illustrated Reviews: Biochemistry (Lippincott Illustrated Reviews C B D Glucokinase and Hexokinase What does the higher Km tell you about the affinity of glucokinase for glucose, as compared to the affinity of hexokinase for glucose? Which enzyme is therefore active even during fasting? Hint: It is the one that can convert even small amounts of glucose into energy HEXOKINASE Which enzyme doesn’t become active until after a highcarbohydrate meal? Hint: It is the one that needs lots of glucose to be activated GLUCOKINASE What do you think is the main role of this enzyme: glycolysis, or glycogenesis? GLYCOGENESIS TRAP DIETARY GLUCOSE (PROVIDING G6P FOR GLYCOGENESIS) Once glycogen stores are full, then glycolysis will be favoured Glucokinase and Hexokinase Time to regroup for a class discussion…if you have finished these slides before we are ready, please look ahead to the following class discussion slides. Glucokinase and Hexokinase Let’s review Reaction? Pathways? Graph? Fasting vs Feeding? A C B D Glucokinase and Hexokinase How does the liver fit into all this? Glucokinase is found here Nutrients absorbed from the intestine go here first Allows glucokinase to convert excess glucose from a meal to glycogen Why can’t other tissues convert excess glucose to glycogen? (Hint: What role does glucose-6-P play?) A lot depends on the tissue (e.g., muscle can make glycogen, glycogen synthesis also occurs in cytosol of tissues.) RBC does NOT synthesize glycogen (not necessary because has readily supply of glucose; same with brain) Adipocytes have hexokinase; adipocytes are designed to store fat; we don’t want to store glycogen there because we already have enough fat as storage https://commons.wikimedia.org/wiki/File:Depiction_of_a_man_suffering_from_fatty_liver.png Glucokinase and Hexokinase So if hexokinase can’t do glycogenesis, what pathway does it feed into? Glycolysis How does this explain its low Km? Does it make sense that glucokinase has a higher Km? Why do you think hexokinase is inhibited by glucose-6-P, but glucokinase is not? Glucokinase and Hexokinase Let’s talk about Vmax High Vmax = high capacity to convert substrate to product Allows glucokinase to phosphorylate lots of glucose to glucose-6-P after a meal, so it can be stored as glycogen Review: What is significant about adding a P to glucose? High Glucose HK pathway used for energy - Runs until what happens ? Glucose Glucose HK Glucose6-P Glycoly sis (-) GK Glycogen esis Glucose6-P GK pathway used for storage - High Vmax: lots of glucose to G-6P - Continues as long as glucose is high. Turns off when glucose is Km: What else is it good for? Km also helps determine enzyme efficiency Kcat Km E+S P measures speed of Product formation once ES has been made measures binding affinity of E and S to make ES ES E+ Enzyme Efficiency Which is a more efficient enzyme: one with a larger or smaller value for Kcat/Km? Kcat Km measures speed of P formation once ES has been made measures binding affinity of E and S to make ES Enzyme Efficiency Not surprisingly, enzymes with very important physiological functions tend be very efficient. What is the general purpose of each of the following very efficient enzymes? Carbonic anhydrase Triose phosphate isomerase Fumarase Acetylcholinesterase Enzyme Inhibition M & M kinetics also aids in the study of enzyme inhibition What are some applications of enzyme inhibition? Physiological feedback mechanisms What example of this did we just see? Clinical therapies Ex: Use of pharmaceuticals to inhibit viral enzymes from replicating in HIV Enzyme Inhibition Types Reversible inhibition Competitive, uncompetitive, noncompetitive Follow M & M kinetics Irreversible inhibition Inhibition of allosteric enzymes (multisubunit) Reversible Enzyme Inhibition: Competitive Competitive inhibition Reversible binding of the inhibitor to the active site of the enzyme I No product S Product https://commons.wikimedia.org/wiki/ Reversible Enzyme Inhibition: Competitive Example Methotrexate (competitive inhibitor) Used for chemotherapy Competitively inhibits the enzyme that helps convert folate (B9) into its coenzyme form FYI: E = dihydrofolate reductase Folate coenzymes normally feed into purine and pyrimidine production – what are these used for? Important when cells are dividing How does methotrexate help prevent the spread of cancer cells? Reversible Enzyme Inhibition: Competitive Competitive inhibition Why doesn’t Vmax change? Does the affinity of the enzyme appear to go up or down in the presence of the inhibitor? Why? Kmapp or Ki https://commons.wikimedia.org/wiki/File:Enzyme_Inhibition_lineweaverburk_plots.gif Reversible Enzyme Inhibition: Uncompetitive Uncompetitive Reversible binding of I to ES Rare Substra te Product formatio n Inhibitor No product formation https://commons.wikimedia.org/wiki/ Reversible Enzyme Inhibition: Uncompetitive Uncompetitive inhibition Is Vmax lower or higher with I? Why? Does the affinity of the enzyme appear to go up or down in the presence of the inhibitor? Why? https://commons.wikimedia.org/wiki/File:Enzyme_Inhibition_lineweaver- Reversible Enzyme Inhibition: Noncompetitive Noncompetitive Reversible binding of I to E or ES Example: Product inhibition G-6-P inhibition of hexokinase Substra te Product formatio n Inhibitor No product formation https://commons.wikimedia.org/wiki/ Reversible Enzyme Inhibition: Noncompetitive Noncompetitive inhibition Is Vmax lower or higher with I? Why? Ki does not change Binding to E appears to lower affinity, binding to ES appears to increase affinity: cancel out! https://commons.wikimedia.org/wiki/File:Enzyme_Inhibition_lineweaver- Practice At Home Which are competitive, uncompetitive, noncompetitive? How do you know? https://commons.wikimedia.org/wiki/File:Enzyme_Inhibition_lineweaverburk_plots.gif Irreversible Enzyme Inhibition Irreversible Occurs when an inhibitor forms a covalent bond with the active site of the enzyme Compare and contrast to competitive inhibition Examples Penicillin Blocks the enzyme required for synthesis of bacterial cell walls Lead poisoning Review: Pb+2 binds to sulfhydryl groups in an enzyme involved in heme synthesis Changes its shape so it no longer functions • How can this lead to anemia? Inhibition of Multi-subunit Allosteric Enzymes Multi-subunit allosteric enzymes Review: What happens when an activator, which could be the substrate, binds? What is the resulting shape of the binding graph? Enzyme What effect would an inhibitor have on the Substrate graph? Slow Faster Fastest Inhibition of Multi-subunit Allosteric Enzymes Sigmoidal graph Inhibitor produces a right shift Enzyme A Enzyme plus Binhibitor C Inhibition of Multi-subunit Allosteric Enzymes Example: phosphofructokinase 1 (PFK1) Review: What does this enzyme do? Rate limiting step of glycolysis Fructose 6 P (ATP ADP) Fructose 1,6 biphosphate Sensitive to ATP levels Binding of F-6-P to one subunit enhances binding of F-6-P to other subunits Creates a sigmoidal graph Allosterically inhibited by ATP What does the term “allosteric inhibition” tell us about where ATP binds when it is acting as an inhibitor vs a substrate? Allosteric inhibitor binds to allosteric site (different than active site) How do you think the Km for these different sites compare? Affinity for active site will be higher (will work at lower concentration for ATP) ATP binds to active and allosteric sites to regulate PFK activity End of Enzyme Kinetics