Regulation of Metabolism FCSN 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

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: 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 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

Hormones, Receptors, and Communication Between Cells 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

Hormones, Receptors, and Communication Between Cells 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

G-protein-linked receptors Hormones, Receptors, and Communication Between Cells Cell-Surface Receptors G-protein-linked receptors Binding of hormone activates an enzyme via a G-protein communication link. The enzymes produces intracellular messengers cAMP diacylglycerol (DAG))

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 Activates or inhibits several enzymes of CHO and Lipid metabolism Inactive form: regulatory+catalytic subunits associated Active form: binding of cAMP disassociates subunits

DAG & IP3 Phosphotidylinositol Signal Transduction Pathway Protein kinase C activated by DAG and calcium Synthesis of DAG and IP3

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 Nitroglycerine slowly produces NO, relaxes cardiac and vascular smooth muscle, reduces angina cAMP activates Protein Kinase G Phosphorylates smooth muscle proteins

cGMP The cGMP Signal Transduction Pathway