Biochem 7_ Enzyme Kinetics PDF

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, Vma...

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 Pre Reading 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 for? ? ? ? ? Application of Km: Assignment Review NAD+ Ethanol NAD+ NADH + H+ Acetaldehyde NADH + H+ Acetate E = aldehyde dehydrogenase Alcohol consumption https://freesvg.org/1455185701 Flushing Back to normal https://commons.wikimedia.org/wiki/File:Smiley_Face.JPG Application of Km and Vmax: Glucokinase • Hexokinase (in 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 Glucokinase and Hexokinase What same reaction (substrate and product) do both hexokinase and glucokinase catalyze? - Glycolysis and glycogenesis The product can be used for one of two glucose metabolism pathways: glucose-6-phosphate • One that breaks down glucose = glycolysis • One that stores glucose = glycogenesis ? ? Product Glucokinase and Hexokinase • Which line in the graph represents: • Glucokinase • Hexokinase A • Which intercepts (A-D) represent: • Vmax • ½ Vmax • Km C B Abali, Emine E; Cline, Susan D; Franklin, David S; Viselli, Susan M. Lippincott Illustrated Reviews: Biochemistry (Lippincott Illustrated Reviews Series) (p. 108). Wolters Kluwer Health. 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? - hexokinase • Hint: It is the one that can convert even small amounts of glucose into energy Which enzyme doesn’t become active until after a high-carbohydrate meal? - glucokinase • Hint: It is the one that needs lots of glucose to be activated • What do you think is the main role of this enzyme: glycolysis, or glycogenesis? 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 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 and will trap glucose as needed • How does this explain its low Km? - low Km = high affinity • Why do you think hexokinase is inhibited by glucose-6-P, but glucokinase is not? Glucokinase and Hexokinase 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 GK HK (-) Glucose-6-P Glycolysis Glycogenesis Glucose-6-P GK pathway used for storage - High Vmax: lots of glucose to G-6-P - Continues as long as glucose is high. Turns off when glucose is low again due to its low affinity Km: What else is it good for? • Km also helps determine enzyme efficiency Kcat Km measures speed of P formation once ES has been made measures binding affinity of E and S to make ES E+S ES E+P 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? example G6P will shut down hexokinase • 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/File:Enzyme_inhibition_schemes.pn Reversible Enzyme Inhibition: Competitive Example: Methotrexate 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? • 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_lineweaver-burk_plots.gif Reversible Enzyme Inhibition: Uncompetitive • Uncompetitive • Reversible binding of I to ES • Rare Substrate Product formation Inhibitor No product formation https://commons.wikimedia.org/wiki/File:Enzyme_inhibition_schemes.pn 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-burk_plots.gif Reversible Enzyme Inhibition: Noncompetitive • Noncompetitive • Reversible binding of I to E or ES • Example: Product inhibition • G-6-P inhibition of hexokinase Substrate Product formation Inhibitor No product formation https://commons.wikimedia.org/wiki/File:Enzyme_inhibition_schemes.pn 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-burk_plots.gif Practice At Home • Which are competitive, uncompetitive, noncompetitive? How do you know? https://commons.wikimedia.org/wiki/File:Enzyme_Inhibition_lineweaver-burk_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? • What effect would an inhibitor have on the graph? Enzyme Substrate Slow Faster Fastest Inhibition of Multi-subunit Allosteric Enzymes • Sigmoidal graph • Inhibitor produces a right shift Enzyme A Enzyme plus B inhibitor C Inhibition of Multi-subunit Allosteric Enzymes Example: Phosphofructokinase 1 (PFK1) • 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? - there has to be 2 sites on the enzyme • There is an active site and an allosteric • How do you think the Km for these different sites compare? End of Enzyme Kinetics