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People would be surprised to learn this about me.
Regulation of Metabolism
NUTR 543
Advanced Nutritional Biochemistry
Dr. David L. Gee
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
Types of Regulatory Mechanisms
• Non-covalent interactions
• Covalent modifications
• Changes in abundance of the
enzyme
Types of Regulatory Mechanisms
• Non-covalent interactions
• Covalent modifications
• Changes in abundance of the
enzyme
Non-covalent Interactions
Substrate 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
Non-covalent Interactions
Allosteric Regulation
• Binding of allosteric effectors at allosteric
sites affect catalytic efficiency of the
enzyme
Non-covalent Interactions
Allosteric Regulation
• Allosteric Activators
– Decrease Km (increases the enzyme binding
affinity)
– Increases Vmax (increases the enzyme catalytic
efficiency)
Non-covalent Interactions
Allosteric Regulation
• Allosteric Inhibitors
– Increases Km (decreases enzyme binding
affinity)
– Decreases Vmax (decreases enzyme catalytic
efficiency)
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
Non-covalent Interactions
Protein-Protein Interactions
• Calmodulin (CALcium MODULted proteIN)
– Binding of Ca++ to calmodulin changes its
shape and allows binding and activation of
certain enzymes
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
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
Calmodulin plays a role in:
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Muscle contraction
Inflammation
Apoptosis
Memory
Immune response….
Metabolism
– Activates phosphorylase kinase
• Stimulates glycogen degradation during exercise
Types of Regulatory Mechanisms
• Non-covalent interactions
• Covalent modifications
• Changes in abundance of the
enzyme
Covalent Regulation of Enzyme Activity
Phosphorylation and Dephosphorylation
• Addition or deletion of phosphate groups to
particular serine, threonine, or tyrosine
residues alter the enzymes activity
Covalent Regulation of Enzyme Activity
Limited Proteolysis
• Specific proteolysis can activate certain
enzymes and proteins (zymogens)
– Digestive enzymes
– Blood clotting proteins
– Peptide hormones (insulin)
Covalent Regulation of Enzyme Activity
Enzyme Cascades
• Enzymes activating enzymes allows for
amplification of a small regulatory signal
Types of Regulatory Mechanisms
• Non-covalent interactions
• Covalent modifications
• Changes in abundance of the
enzyme
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
Hormones, Receptors, and
Communication Between Cells
• Hormones
– chemical signals that coordinate metabolism
• Hormone Receptors
– Target tissues
– Specific binding
– Types
• Intracellular receptors
• Cell-surface receptors
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
Hormones, Receptors, and Communication Between Cells
Intracellular Receptors
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
Cell Surface Receptors
Ligand-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
Cell-Surface Receptors
Catalytic Receptors
• Binding of hormone activates tyrosine kinase on
receptor which phosphorylates certain cellular
proteins
• Insulin receptor is a catalytic receptor with TYR
Kinase activity
Cell-Surface Receptors
G 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
Intracellular Messengers:
Signal Transduction Pathways
• Cyclic AMP (cAMP)
• Diacylglycerol (DAG) &
Inositol Triphosphate (IP3)
• Cyclic GMP (cGMP)
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
G Proteins
• G proteins are
trimers
– Three protein units
• Alpha
• Beta
• gamma
• 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
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)
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
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
Clinical Case
• 25 y.o. female vacationing in Costa Rica
• Severe diarrhea, nearly comatose
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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
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
Cholera
mechanism 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
Clinical Case
• V. cholerae toxin affects alphaS 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
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 ?
Intracellular Messengers:
Signal Transduction Pathways
• Cyclic AMP (cAMP)
• Diacylglycerol (DAG) &
Inositol Triphosphate (IP3)
• Cyclic GMP (cGMP)
DAG & IP3
Phosphotidylinositol Signal Transduction Pathway
• Hormone activation of phospholipase C
– Via Gp protein
• Phospholipase C hydrolyzes membrane
phospholipids (phosphotidyl inositol) to produce
DAG and IP3
DAG & IP3
Phosphotidylinositol Signal Transduction Pathway
• IP3 stimulates release of Ca from ER
• Protein kinase C activated by DAG and
calcium
Intracellular Messengers:
Signal Transduction Pathways
• Cyclic AMP (cAMP)
• Diacylglycerol (DAG) & Inositol
Triphosphate (IP3)
• Cyclic GMP (cGMP)
cGMP
The 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
cGMP
The 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
cGMP
The cGMP Signal Transduction Pathway