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 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

Non-covalent InteractionsAllosteric Regulation

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

Non-covalent InteractionsAllosteric Regulation

• Allosteric Activators– Decrease Km (increases the enzyme binding

affinity)– Increases Vmax (increases the enzyme catalytic

efficiency)

Non-covalent InteractionsAllosteric 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 InteractionsProtein-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 ActivityPhosphorylation and Dephosphorylation

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

Covalent Regulation of Enzyme ActivityLimited Proteolysis

• Specific proteolysis can activate certain enzymes and proteins (zymogens)– Digestive enzymes– Blood clotting proteins– Peptide hormones (insulin)

Covalent Regulation of Enzyme ActivityEnzyme 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

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

• 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

DAG & IP3

Phosphotidylinositol Signal Transduction Pathway

• Protein kinase C activated by DAG and calcium

• Synthesis of DAG and IP3

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

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

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

• cAMP activates Protein Kinase G– Phosphorylates smooth muscle proteins

cGMPThe cGMP Signal Transduction Pathway