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Introductions PowerPoint PPT Presentation


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Introductions. Name Email address Favorite type of food Least favorite food Magazine you read most often All-time favorite movie Concert/performance you’d most want to see Favorite sport to play / favorite sport to watch People would be surprised to learn this about me.

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Introductions

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Introductions l.jpg

Introductions

  • Name

  • Email address

  • Favorite type of food

  • Least favorite food

  • Magazine you read most often

  • All-time favorite movie

  • Concert/performance you’d most want to see

  • Favorite sport to play / favorite sport to watch

  • People would be surprised to learn this about me.


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Regulation of Metabolism

NUTR 543

Advanced Nutritional Biochemistry

Dr. David L. Gee


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Characteristics of Regulatory Enzymes

  • Catalyze a rate-limiting step

  • Catalyze a committed step

    • Early step unique to a pathway

    • Irreversible step

      • Requires energy

      • Often results in a phosphorylated compound


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Types of Regulatory Mechanisms

  • Non-covalent interactions

  • Covalent modifications

  • Changes in abundance of the enzyme


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Types of Regulatory Mechanisms

  • Non-covalent interactions

  • Covalent modifications

  • Changes in abundance of the enzyme


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Non-covalent InteractionsSubstrate availability

  • Non-regulatory enzymes generally exhibit hyperbolic kinetics (Michaelis-Menton)

    • At low substrate concentration, reaction rate proportional to substrate concentration

  • Regulatory enzymes generally exhibit sigmoidal kinetics (positive cooperativity)

    • Changes of substrate concentrations at normal physiological levels greatly alter reaction rate


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    Non-covalent InteractionsAllosteric Regulation

    • Binding of allosteric effectors at allosteric sites affect catalytic efficiency of the enzyme


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    Non-covalent InteractionsAllosteric Regulation

    • Allosteric Activators

      • Decrease Km (increases the enzyme binding affinity)

      • Increases Vmax (increases the enzyme catalytic efficiency)


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    Non-covalent InteractionsAllosteric Regulation

    • Allosteric Inhibitors

      • Increases Km (decreases enzyme binding affinity)

      • Decreases Vmax (decreases enzyme catalytic efficiency)


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    Molecues that act as allosteric effectors

    • End products of pathways

      • Feedback inhibition

    • Substrates of pathways

      • Feed-forward activators

    • Indicators of Energy Status

      • ATP/ADP/AMP

      • NAD/NADH

      • Citrate & acetyl CoA


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    Non-covalent InteractionsProtein-Protein Interactions

    • Calmodulin (CALcium MODULted proteIN)

      • Binding of Ca++ to calmodulin changes its shape and allows binding and activation of certain enzymes


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    Binding of calcium to Calmodulin changes the shape of the protein

    Unbound Calmodulin on left

    Calcium bound Calmodulin on right. Stars indicate exposed non-polar ‘grooves’ that non-covalently binds proteins


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    Calmodulin

    • Extracellular [Ca] = 5 mM

    • Intracellular [Ca] = 10-4 mM

      • Most of Ca bound inside cells

      • Bound Ca can be released by hormonal action, nerve innervation, light, ….

      • Released Ca binds to Calmodulin which activates a large number of proteins


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    Calmodulin plays a role in:

    • Muscle contraction

    • Inflammation

    • Apoptosis

    • Memory

    • Immune response….

    • Metabolism

      • Activates phosphorylase kinase

        • Stimulates glycogen degradation during exercise


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    Types of Regulatory Mechanisms

    • Non-covalent interactions

    • Covalent modifications

    • Changes in abundance of the enzyme


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    Covalent Regulation of Enzyme ActivityPhosphorylation and Dephosphorylation

    • Addition or deletion of phosphate groups to particular serine, threonine, or tyrosine residues alter the enzymes activity


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    Covalent Regulation of Enzyme ActivityLimited Proteolysis

    • Specific proteolysis can activate certain enzymes and proteins (zymogens)

      • Digestive enzymes

      • Blood clotting proteins

      • Peptide hormones (insulin)


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    Covalent Regulation of Enzyme ActivityEnzyme Cascades

    • Enzymes activating enzymes allows for amplification of a small regulatory signal


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    Types of Regulatory Mechanisms

    • Non-covalent interactions

    • Covalent modifications

    • Changes in abundance of the enzyme


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    Changes in Enzyme Abundance

    • Inducible vs Constitutive Enzymes

    • Induction is caused by increases in rate of gene transcription.

      • Hormones activate transcriptional factors

        • Increase synthesis of specific mRNA

        • Increase synthesis of specific enzymes


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    Hormones, Receptors, and Communication Between Cells

    • Hormones

      • chemical signals that coordinate metabolism

    • Hormone Receptors

      • Target tissues

      • Specific binding

      • Types

        • Intracellular receptors

        • Cell-surface receptors


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    Hormones, Receptors, and Communication Between Cells

    • Intracellular receptors

    • lipid soluble hormones

      • Steroid hormones, vitamin D, retinoids, thyroxine

  • Bind to intracellular protein receptors

    • This binds to regulatory elements by a gene

    • Alters the rate of gene transcription

      • Induces or represses gene transcription


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    Hormones, Receptors, and Communication Between CellsIntracellular Receptors


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    Hormones, Receptors, and Communication Between Cells

    • Cell-surface receptors

      • Water soluble hormones

        • Peptide hormones (insulin), catecholamines, neurotransmitters

    • Three class of cell-surface receptors

      • Ligand-Gated Receptors

      • Catalytic Receptors

      • G Protein-linked Receptors


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    Cell Surface ReceptorsLigand-Gated Receptors

    • Binding of a ligand (often a neurotransmitter) affects flow of ions in/out of cell

    • Gamma-amino butyric acid (GABA) binds and opens chloride channels in the brain

      • Valium (anti-anxiety drug) reduces the amount of GABA required to open the chloride channels


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    Cell-Surface ReceptorsCatalytic Receptors

    • Binding of hormone activates tyrosine kinase on receptor which phosphorylates certain cellular proteins

    • Insulin receptor is a catalytic receptor with TYR Kinase activity


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    Cell-Surface ReceptorsG Protein-Linked Receptors

    • Binding of hormone activates an enzyme via a G-protein communication link.

    • The enzymes produces intracellular messengers

      • Signal transduction

      • Second messengers activate protein kinases


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    Intracellular Messengers:Signal Transduction Pathways

    • Cyclic AMP (cAMP)

    • Diacylglycerol (DAG) & Inositol Triphosphate (IP3)

    • Cyclic GMP (cGMP)


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    G-Protein-Linked Receptors:The cAMP Signal Transduction Pathway

    • Two types of G-Proteins

    • Stimulating G protein (Gs)

      • Activate adenylate cyclase

    • Inhibitory G proteins (Gi)

      • Inhibit adenylate cyclase


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    G Proteins

    • G proteins are trimers

      • Three protein units

        • Alpha

        • Beta

        • gamma


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    • Alpha proteins are different in Gs and Gi

      • Both have GTPase activity

      • Alpha proteins modify adenylate cyclase activity

        • AC stimulated by Alpha(s) when activated by a hormone

        • AC Inhibited by Alpha(I) when activated by other hormones


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    Family of G Proteins

    • Binding of hormones to receptors causes:

      • GTP to displace GDP

      • Dissociation of alpha protein from beta and gamma subunits

      • activation of the alpha protein

      • Inhibition or activation of adenylate cyclase

      • GTPase gradually degrades GTP and inactivates the alpha protein effect (clock)


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    The cAMP Signal Transduction Pathway

    • cAMP – intracellular messenger

      • Elevated cAMP can either activate or inhibit regulatory enzymes

        • cAMP activates glycogen degradation

        • cAMP inhibits glycogen synthesis

    • [cAMP] affected by rates of synthesis and degradation

      • Synthesis by adenylate cyclase

      • Degradation by phosphodiesterase

        • Stimulated by insulin

        • Inhibited by caffeine


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    What does cAMP do?Activation of Protein Kinase A by cAMP

    • Protein kinase A

      • Activates or inhibits several enzymes of CHO and Lipid metabolism

      • Inactive form: regulatory+catalytic subunits associated

      • Active form: binding of cAMP disassociates subunits


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    Clinical Case

    • 25 y.o. female vacationing in Costa Rica

    • Severe diarrhea, nearly comatose

      • Diarrhea resulting in fluid loss of ~800 ml/hr

      • Hypotensive (75/50)

      • Metabolic acidosis , low bicarbonate

      • Stool sample contained Vibrio cholerae

    • IV administration of fluids, tetracycline

      • Patient improves rapidly


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    Cholera background information

    • Severe and rapid diarrheal disease

      • Caused by Vibrio cholerae

      • Commonly shock after 4-12 hrs after first symptoms, death 18 hrs – several days without rehydration therapy (subject can lose up to 20 liters of fluids)

      • Source is commonly contaminated water


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    Choleramechanism of action

    • V. cholarae produces protein that attaches to intestinal epithelial cells

      • Delivery subunit B (blue) facilitates entry of subunit A into cell

    • Subunit A catalyzes ADP-ribosylation of the alpha-s subunit of Gs-protein


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    Clinical Case

    • V. cholerae toxin affects alpha-S subunit

      • Inactivates GTP’ase

      • Alpha-S subunit permanently active

    • Stimulates adenylate cyclase

      • Overproduces cAMP

      • stimulates protein kinase

      • Phosphorylation of membrane ion transport proteins – massive losses of Na, Cl, K, HCO3


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    Hypothetical link to cystic fibrosis

    • Cystic fibrosis characterized by

      • Salty sweat

      • Very thick mucous

    • Homozygous genetic defect to chloride transport to mucous

      • Decreased chloride results in less water following due to osmosis, leading to thicker mucous

    • Heterozygous mutation (normal mucous) has transport protein resistant to effects of cholera toxin ?


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    Intracellular Messengers:Signal Transduction Pathways

    • Cyclic AMP (cAMP)

    • Diacylglycerol (DAG) & Inositol Triphosphate (IP3)

    • Cyclic GMP (cGMP)


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    DAG & IP3Phosphotidylinositol Signal Transduction Pathway

    • Hormone activation of phospholipase C

      • Via Gp protein

    • Phospholipase C hydrolyzes membrane phospholipids (phosphotidyl inositol) to produce DAG and IP3


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    DAG & IP3Phosphotidylinositol Signal Transduction Pathway

    • IP3 stimulates release of Ca from ER

    • Protein kinase C activated by DAG and calcium


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    Intracellular Messengers:Signal Transduction Pathways

    • Cyclic AMP (cAMP)

    • Diacylglycerol (DAG) & Inositol Triphosphate (IP3)

    • Cyclic GMP (cGMP)


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    cGMPThe cGMP Signal Transduction Pathway

    • cGMP effects:

    • lowering of blood pressure & decreasing CHD risk

      • Relaxation of cardiac muscle

      • Vasodilation of vascular smooth muscle

      • Increased excretion of sodium and water by kidney

      • Decreased aggregation by platelet cells


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    cGMPThe cGMP Signal Transduction Pathway

    • Two forms of guanylate cyclase

    • Membrane-bound

      • Activated by ANF (atrial natriuretic factor)

        • ANF released when BP elevated

  • Cytosolic

    • Activated by nitric oxide

    • NO produced from arginine by NO synthase (activated by Ca)

      • Nitroglycerine slowly produces NO, relaxes cardiac and vascular smooth muscle, reduces angina

  • cGMP activates Protein Kinase G

    • Phosphorylates smooth muscle proteins


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    cGMPThe cGMP Signal Transduction Pathway


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