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Fine-Tuning Enzyme Activity in Cells: Mechanisms and Regulation

Explore how cells finely regulate enzyme activity through allosteric regulation, covalent modification, and feedback mechanisms. Learn about varied enzyme types and control methods.

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Fine-Tuning Enzyme Activity in Cells: Mechanisms and Regulation

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  1. How does a cell fine tune the activity of its enzymes? 9/12 and 9/14 How do cells control enzyme activity? • What is allosteric regulation? Can it stimulate or inhibit? • How does allosteric regulation control glycogen synthesis? • How can the products of a reaction pathway affect the enzymes that lead substrate into the pathway? • What is the most common type of covalent modification? • How can proteolytic cleavage be used to regulate enzyme function? • What is a ribozyme? • What are the functions of a plasma membrane?

  2. Assignment Due at class Monday: CH 5Que #: 5-6, 5-7 CH6 Que#: 6-1, 6-4, 6-5 and 6-6Copy of answer key is located in library at info desk for short term check out. 10 pts

  3. What are the ten different types of enzyme? 1) Hydrolase: cleaves substrate by adding H2O 2) Nuclease: cleaves adjacent nucleotides with H2O 3) Protease: cleaves peptide bonds with H2O 4) Synthase: makes complex molecule from precursors 5) Isomerase: rearranges functional group on substrate 6) Polymerase: synthesize DNA or RNA from nucleotides 7) Kinase: adds a phosphate to target 8) Phosphatase: removes phosphate from target 9) Oxido-Reductase (Dehydrogenase): one molecule is oxidized and one reduced 10) ATPase: hydrolyzes ATP to ADP to generate energy Some overlap may exist between types!

  4. Lets Review the ways to control enzyme activity:Which are available to prokaryotes? • Enzyme Production/Destruction • Via DNA/mRNA transcription/translation: • Release into the blood/target • Failure to replace it after turnover • Temperature and pH: • Allosteric Inhibition/Activation: • Catalytic/Regulatory subunits- • Covalent Modification: • Phosphorylation/Dephosphorylation- • Protein Kinase/Protein Phosphatase- • Proteolytic Cleavage: • Autocatalytic Activity:

  5. How do cells control enzyme activity? • Some produce E only when needed (think prokaryotes!) • Some let E turn-over when no longer used • Some modify activity of E to meet needs at the time FeedBack-Inhibition: Sometimes a product inhibits E to prevent excess/wasteful production of product FeedForward-Stimulation: Sometimes a product stimulates E to promote greater production of product • Classic Pattern: molecule exerting effect on entry enzyme is often the last molecule produced in the pathway! • A  BCDE but E-inhibits/stimulates entry to pathway at the AB-Enzyme

  6. Allosteric regulation occurs when an enzyme exists in one of two forms (fast/slow) and form taken is dependent upon the binding of a regulator at a site other than the active site. Feed Back Inhibition/ Allosteric Inhibition: • Observation about Glycolysis: • G + ATP  G-6-P + ADP (Hexokinase) • Committed first step of glycolysis! • G-6-P can non-permanently bind Hexokinase at an allosteric site and modify its conformation! • The Two Forms of Hexokinase: • Fast (no G-6-P) vs Slow(when G-6-P Present!) • +Km or +Vm -Km or -Vm • Why is this useful to a cell? What happens after a meal in a diabetics skeletal muscle cell?

  7. Allosteric regulation: enzyme exists in one of two forms (fast/slow) and form taken is dependent upon binding of a regulator at a site other than active site. Feed FORWARD Stimulation/ Allosteric activation: Observation regarding Glycolysis: G-6-PF-6-PF-1,6-PPEP(Pyruvate Kinase)Pyruvate • How do we speed up the system at the END based on the presence of the entry of substrate at the START of system? Translated: how does a cell speed up energy use when there is plenty of energy available? Early product F-1,6-P  Stimulates PK activity at last step! • The Two Forms of pyruvate kinase: • Fast vs Slow • When do you want these to be fast/slow? • Signal? F-1,6-P is allosteric regulator promoting FAST PK-form

  8. Allosteric Regulation of an Enzyme (protein): Molecule binds to non-active site! Typically binding to dimers or larger! Often regulator is product in pathway!

  9. Cells also regulate activity by covalently modifying enzymes by covalent modification, typically adding/removing PO4-groups! Glycogen synthase and Glycogen phosphorylase are reciprocally regulated classics in this regard!

  10. Glycogen is broken down to G-1-P either slowly or rapidly depending on the activity state of Glycogen Phosphorylase a!How do we know when to phosphorylate?

  11. In this case phosphorylase kinase (a cAMP dependent protein kinase) is able to phosphorylate its target glycogen phosphorylase a when the hormone glucagon causes the cell to accumulate cyclic AMP. cAMP made when cell is hungrybinds to.. cAMP-dependent Protein kinase Phosphorylates targetactivation of enzyme!

  12. Proteolytic cleavage can also be an important signal for enzyme activation. This way an enzyme is not turned on until it is in the correct location in body/cell. Applications in the intestine and in Emphysema • Pancreatic Enzymes: • Zymogens: In active protein products • Pancreatic Zymogens: • Typsinogen, Chymotrypsinogen, Procarboxylpeptidase • Intestinal Enterokinase only cleaves Trypsinogen! • Trypsinogen activates remaining zymogens! • The liver also produces a plasma protein called: • Alpha-1-antitrypsin (irreversible competitive inhibitor) to prevent inappropriate self-digestion of elastin and other connective fibers.

  13. What happens to the balance of trypsin secretion and activity in a chronic smoker? An irreversible competitive inhibitor is lost! Trypsin is activated and digests collagen and elastin! Lung loses all elasticity! 1) Smoking oxidizes a methionine residue on inhibitor! • R-CH2-CH2-S-Ch3  R-CH2-CH2-SO-CH3 2)Oxidized product can’t inhibit enzyme! • Damaged lungs are rich in neutrophils 3) Trypsin is released and elastin destroyed! 4) Lungs must now forcibly contract to inhale AND exhale! 5) Our old male cadaver in the AP lab had this! • The pancreas produces a similar protein inhibitor to protect itself against any trypsin that has its regulatory tail removed accidentally!

  14. Ribozymes (RNA) are the least common enzyme (they do not consist of protein). They are typically autocatalytic in function.

  15. More about enzymes and kinetics • More about M-M kinetics hyperbolic curves, and approximations • LB plots, straight lines, and exact predictions • Why is it valuable to know km, Vm, V1/2, etc with respect to cell culture or drug metabolism by cells? • Drugs as non-competitive and competitive inhibitors in cells • LAST MATERIAL FOR the First Cell Bio Lecture EXAM: • Michealis-Menton Kinetics will be on exam • Lineweaver-Burke plots will not be on exam • 50 points (bring scantron) Exam will be Ch 1-3(mostly review) and 4-6 Exam will emphasize what is in the notes Textbook should help understand the notes and is important, especially the problems in the back of the chapters.

  16. The active site is where the substrate binds to the enzyme. What are the six features of an active site? • 1) AS is small part of total! • 2) AS spatial in 3-D! • 3) AS hold substrate by using its specific charges on its amino acids to attract substrate! • Ionic, Hydrogen, Van der Waals Forces • Covalent bonds only at transition state! • 4) AS located in peptide cleft! • 5) Substrate/Cleft perfectly align! • 6) Substrate/Enzyme binding causes induced fit at site! • Induced fit result in change to shape of entire enzyme!

  17. Competitive and Non-competitive Inhibitors bind to different sites!

  18. Michealis-Menton Enzyme Kinetics refers to how fast (quantity/time) an enzyme catalyzes a particular chemical reaction! There are special rules that apply inside a cell! • Velocity: [P]/time! • Initial Reaction Velocity: speed at start! • Saturation: When an enzyme can’t handle ANY more substrate per time,,,all active sites are filled up! • Vmax: Maximum theoretical velocity!=traffic jam • ½ Vmax: Half of maximum, typically organisms let their enzymes function near this value. • Michaelis-Menton Kinetics: refers to the fact that an the substrate MUST bind to the enzyme BEFORE becoming product! This creates a special type of Keq Equation!

  19. Normal: SubstrateProducts Keq=[p]/[s]M-M Kinetics: Substrate + Enz >Enz-S<Product + EnzymeThere are now 4 reaction directions to consider in this reversible reaction! Keq is now called Km or a Michaelis-Menton Constant!Each enzyme has a specific Km value associated with it!

  20. Km Values are important to cell biologists because they let us know the health/unhealth of a cell given a set of conditions and let us predict cell function/adaptability! • How much toxin/time can the cells of a weed degrade? When does farmer Pat need to reapply herbicide and how much? • How much lactose can Pat digest/time? How much milk can Pat drink without getting diarrhea, assuming lactose insufficiency? • How much chemotherapy drug can be destroyed/time by the hapatocytes of a healthy or unhealthy liver? When does Bob need to take more or less? What is the monetary cost? • How much glucose can be metabolized over time by the myocytes of an Olympic sprinter? How much of his illegal drug can be metabolized? Will he beat the test?

  21. Competitive inhibitors change Km!-MMBecause fewer sites are available to the substrate!Non-Competitive Inhibitors change Vm!-MM Because fewer enzymes are available to the substrate!

  22. Competitive and Non-competitive Inhibitors bind to different sites!

  23. Irreversible Enzyme Inhibitors: DFP is similar to the Sarin Nerve Gas for use on humans in warfare

  24. Lineweaver-Burke Plots prepresent a double reciprocal system for comparing reaction velocity to substrate concentration.Competitive inhibitors change 1/Km!-LBBecause fewer sites are available to the substrate!Non-Competitive Inhibitors change 1/Vm!-LB Because fewer enzymes are available to the substrate!

  25. Consider these problems:1) With respect to 1/Km and 1/Vmax, which of these two Lineweaver-Burke Plots (See below) is from a system experiencing Competitive and Non-Competitive Inhibition? 2) If you need the product of a reaction that is irreversibly inhibited (I.e. Sarin Nerve Gas effect on acetylcholinesterasedruignwarefare), what must the cells of your body do to survive in the presence of the irreversible inhibitor?

  26. LB Plot with inhibitorsIs A or B competitive and non-competitive?

  27. Figure From: Maher MA, Mataczynski H, Stefaniak HM, Wilson T. Cranberry juice induces nitric oxide-dependent vasodilation in vitro and in vivo and its infusion transiently reduces blood pressure in anaesthetized rats. J Med Foods 2000;3:141-147.

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