BMS2021 Topic A: Regulation of Metabolism - Week 1 Notes

Topic A: Regulation of Metabolism

Topic Overview

Week 1: Introduction to Metabolic Regulation
- Glucose Homeostasis
- Background: The Pathways
  - Glycolysis
  - Gluconeogenesis
  - Glycogenesis & Glycogenolysis
- How metabolic processes are regulated
  - Amount of enzyme
  - Activity of enzyme
- Regulation of Glucose Homeostasis
- Regulation of Glycolysis & Gluconeogenesis
- Regulation of Glycogenesis & Glycogenolysis
Week 2: Regulation of Metabolism in Fasting and Starvation states
- Nitrogen metabolism
- How our bodies use nitrogen
- The urea cycle & nitrogen balance
- Metabolism in fasting and starvation states
- The players: tissues, fuels, and hormones
- The states: Fed, fasting, starvation, and diabetes

Topic Introduction: What is Metabolism?

Definition: The entire set of enzyme-catalyzed transformations of organic molecules in living cells; the sum of anabolism and catabolism.
Enzymes:
- Proteins that are substrate-specific.
- Increase the rate of a reaction.
- Initiate a change and accelerate reactions.
Organic Molecules:
- Carbon-containing compounds include carbohydrates, fats, and proteins.
Anabolism:
- Synthesis processes that are energy-requiring.
Catabolism:
- Breakdown processes that are energy-releasing.

Learning Outcomes

Discuss the importance of the regulation of cellular metabolism.
Describe the general principles of regulation of enzyme amount and activity.
Identify the key mechanisms of enzyme regulation in relation to metabolism.
Apply the principles of regulation to glucose homeostasis.
Explain the significance of glycolysis, gluconeogenesis, glycogenesis, and glycogenolysis in the regulation of glucose homeostasis.
Identify and describe the regulatory mechanisms involved in glycolysis, gluconeogenesis, glycogenesis, and glycogenolysis.

Hormonal Regulation

Key Hormones: Insulin, glucagon, and epinephrine.
Mechanism:
- Chemical signals released into the blood.
- Only target cells respond to a given hormone.
- Hormones combine with specific receptor proteins.
- Hormones are quickly eliminated from the blood.
Control: Can control both the amount and the activity of an enzyme.
Comparison:
- Hormonal control (intercellular) is slower than regulation by allosteric activation/inhibition and covalent modification (intracellular).
- Effects can result in 10-20 fold increases in enzyme activity.
Release Triggers:
- Insulin: Released when glucose concentration is too high.
- Glucagon: Released when glucose concentration is too low.
- Epinephrine: Released to prepare muscles, lungs, and heart for a burst of activity.

How Metabolic Processes Are Regulated

Two main factors:
1. Amount of enzyme
2. Activity of enzyme

Amount of Enzyme

1. Lifespan of Enzyme

All proteins have finite lifespans.
Different proteins in the same tissue have very different half-lives (less than an hour to about a week for liver enzymes).
Stability correlates with the sequence at the N-terminus.
Some proteins are as old as you are (e.g., Crystallins in the eye lens).
Table of Average Half-Life of Proteins in Mammalian Tissues:
- Liver: 0.9 days
- Kidney: 1.7 days
- Heart: 4.1 days
- Brain: 4.6 days
- Muscle: 10.7 days

2. Constitutive Enzymes

Long lifespans: days to months.
Rate of synthesis = rate of degradation.
Required in constant concentrations.
Examples: Glycolytic enzymes, citric acid cycle enzymes.

3. Inducible/Repressible Enzymes

Short lifespans, synthesized only when required.
Rate of synthesis and degradation can be increased or decreased.
Examples: Hormones, growth factors.

4. Induced or Repressed Synthesis

The synthesis of an enzyme can be controlled, either increasing its expression or decreasing it.
Example: Induced by insulin
- Hexokinase II & glucokinase (Glu → G-6-P): ↑ Glycolysis
- liver phosphofructokinase (PFK-1): ↑ Glycolysis
Example: Repressed by insulin
- PEP Carboxykinase
- Glucose 6-phosphatase: Gluconeogenic enzymes therefore ↓GNG
- Repression: FOXO1 is a transcription factor that is degraded in response to insulin signaling.

5. Compartmentalization

Glucose-6-phosphatase:
- G-6-P → Glucose + Pi
- In gluconeogenic tissues (liver, kidney) but not in glycolytic tissues (muscle, brain, fat).
  Red blood cells do not contain mitochondria, therefore do not have the enzymes for the:
- Citric acid cycle
- Electron transport chain
Hexokinase:
- Sequestered to the nucleus until conditions favor glycolysis in the liver (high [glucose]).

6. Exist as Isozymes

Multiple forms of an enzyme that catalyze the same reaction but differ in amino acid sequence, substrate affinity, V_{max}, and/or regulatory properties.
May exist within a single cell/tissue.
Are often the products of different genes.
Catalyze the same reaction but have different primary structures.
Example: Hexokinase
- Reaction: Phosphorylation of glucose, the first step of Glycolysis: ATP + glucose \longrightarrow Glu-6-P + ADP
- Four iso-enzymes of hexokinase:
  - Hexokinase I, II, and III (Muscle): K_m for glucose = 0.04 mM (Low)
  - Hexokinase IV (Glucokinase) (Liver): K_m for glucose ~10 mM (High)
  - Blood [glucose] is about 5.0 mM
  - Liver [glucose] is about 5.0 mM
  - Muscle [glucose] is <0.5 mM.
- Other examples include pyruvate kinase and lactate dehydrogenase.

Activity of Enzyme

1. Concentration of Substrate(s)

The rate of a reaction depends on the concentration of substrates.
The rate is more sensitive to concentration at low concentrations.
- Chemical kinetics: Frequency of substrate meeting the enzyme matters.
The rate becomes insensitive at high substrate concentrations.
- The enzyme is nearly saturated with substrate.
Example: Cellular transport of glucose across membranes
- Facilitated transport: Glucose transporter GLUT1-5
  - Concentration of glucose in blood plasma ~ 4.5 mM
  - Concentration of glucose in cytoplasm much lower
  - Glucose enters cells through specific transporters
  - Glucose uptake by the brain and red blood cells is insulin-independent.
  - Glucose uptake by muscle and adipose tissue is insulin-dependent.
- Model for glucose transport into red blood cells by GLUT-1.
- Glucose uptake by muscle and adipose tissue is insulin-dependent
  - Insulin stimulates translocation of GLUT-4 glucose transporters to the surface of myocytes (synthesizing glycogen) and adipocytes (synthesizing triacylglycerols).
  - Results in an increase in glucose uptake to 15-fold or more.
  - In type I diabetes: there is no insulin released and therefore no mobilization of GLUT-4.

2. Allosteric Modulation

REVERSIBLE, NON-COVALENT binding of a modulator at a site other than the active site.
Allosteric enzymes:
- Have a separate binding site for their modulators (inhibitors or activators).
- Have quaternary structure and are composed of subunits.
- The subunits can adopt more than one conformation.
- Binding of substrate occurs more readily to one conformation.
- Binding of a modulator brings about a conformational change in the enzyme.
- C = Catalytic subunit
- R = Regulatory subunit

3. Phosphorylation

Addition of a phosphate group.
- Covalent modification.
Phosphorylation is catalyzed by protein kinases.
Dephosphorylation is catalyzed by protein phosphatases or can be spontaneous.
Typically, proteins are phosphorylated on the hydroxyl groups of Ser, Thr, or Tyr.
Phosphorylation may activate or inactivate an enzyme.
Think of it as a switch.

4. Regulatory Molecules (Coenzymes/Proteins)

Cells contain limited concentrations of coenzymes such as:
- NAD^+ and NADH
- NADP^+ and NADPH
- ATP, ADP, and AMP
- Acetyl-S-CoA
In cells, [NAD^+] + [NADH] = 0.5mM
Binding of regulatory protein subunits affects specificity.
In this example, two different regulatory subunits exist.
Creates different substrate-binding sites.

Regulation of Glycolysis and Gluconeogenesis

Glycolysis and Gluconeogenesis
- Key Enzymes and Regulatory Steps
  - Hexokinase/Glucose 6-phosphatase
  - Phosphofructokinase-1/Fructose 1,6-bisphosphatase-1
  - Pyruvate kinase/Pyruvate carboxylase/PEP carboxykinase

1. Bypass Reaction 3: Futile Cycle 1

Glucose 6-phosphatase.
- Present in hepatocytes (liver cells) but not in muscle cells or many other cell types.
- Gluconeogenesis prevented in cells without this enzyme.
Hexokinase.
- Present in all cells with Glycolytic activity (nearly all cell types).
- Isozymes regulated differently.
Four isozymes for hexokinase exist (I to IV).
Different isozymes exist in different tissues.
Isozymes perform the same reaction but can be regulated differently.
Hexokinase I & II
- Found in muscle cells.
- Allosterically inhibited by their product, Glucose 6-phosphate.
- High affinity for Glucose (Low K_m).
Hexokinase IV (also called Glucokinase)
- Found in the liver.
- Low affinity for Glucose (High K_m).
- Inhibited by a nuclear binding protein.

2. Bypass Reaction 2: Futile Cycle 2

Phosphofructokinase-1 (PFK-1)
- Allosterically regulated
  - Inhibits Phosphofructokinase-1 when [ATP] is high.
  - Relieves the inhibition of Phosphofructokinase-1 by ATP when [AMP] and [ADP] are high. Therefore, Activates.
  - Increases the affinity of PFK-1 for Fructose 6-phosphate, decreases its affinity for ATP and Citrate.
  - Increases the inhibitory action of ATP
Fructose 1,6-bisphosphatase-1
- Allosterically regulated
  - Inhibited by increased AMP – a byproduct of ATP consumption.
  - Inhibited by Fructose 2,6-bisphosphate
  - Example of reciprocal regulation.

3. Bypass Reaction 1: Futile Cycle 3

Pyruvate kinase
- Three isozymes (L= liver, M = Muscle).
- Allosterically activated by fructose-1,6-bisphosphate: high flow through glycolysis.
- Allosterically inhibited by signs of abundant energy supply (all tissues).
  - ATP
  - Acetyl-CoA and long-chain fatty acids
  - Alanine (enough amino acids).

Regulation of Glycogenesis and Glycogenolysis

1. General Information

Genesis = synthesis | Lysis = breakdown
Muscle Glycogen:
- Provides a quick source of energy for either aerobic or anaerobic metabolism.
- Can be used up in less than one hour during vigorous activity.
Liver Glycogen:
- Serves as a reservoir of glucose for other tissues when dietary glucose is not available. This is especially important for the brain.
- Can be depleted in 12-24 hours.
The general mechanism for storing and mobilizing glycogen is the same in the muscle and the liver, but the enzymes differ reflecting the different roles of glycogen in the two tissues. Relevant Enzymes:
- Phosphoglucomutase
- Hexokinase
- UDP-glucose pyrophosphorylase
  Regulation is controlled at multiple levels:
- at the hormonal level
- at the enzyme level
- allosteric regulation and
- covalent regulation of glycogen synthase and glycogen phosphorylase

2. Regulation of Glycogen Synthesis

Controlled through regulating glycogen synthase
- Insulin-signaling pathway
  - increases glucose import into muscle
  - stimulates the activity of muscle hexokinase
  - activates glycogen synthase
  - Increased hexokinase activity enables activation of glucose

3. Regulation of Glycogen Breakdown

Controlled through regulating glycogen phosphorylase
- Glucagon/Epinephrine signaling pathway (hormonal regulation)
  - Starts phosphorylation cascade via intracellular signaling molecule cAMP (allosteric regulation)
  - activates glycogen phosphorylase
- Glycogen phosphorylase:
  - An allosteric enzyme
  - Regulated by covalent modification (reversible phosphorylation)
  - Glycogen phosphorylase cleaves glucose residues off glycogen, generating glucose-1-phosphate
  - Tissue-specific
Glycogen phosphorylase in the liver is inhibited by glucose
Hormonal control is tissue-specific

4. Coordinated regulation of glycogen synthesis and breakdown

Blood glucose homeostasis