Intermediary Metabolism Overview

• The presentation focuses on intermediary metabolism, presented by Prof Alisa Phulukdaree.
• It will cover week 1 and 2 of the broad overview.

Topics Covered

• Homeostasis
• Enzymes
• Carbohydrate metabolism
• Lipid metabolism
• Ketone metabolism
• Protein metabolism
• Inborn errors of metabolism
• Vitamins & deficiencies
• Integrated metabolism
• Insulin & glucagon

Homeostasis

• Denotes stability, balance, or equilibrium within cells or the entire body.
• It requires adjustments of internal and external conditions.
• Homeostatic regulation is part of this process.
• Continuous adjustments are made to meet the 'Set Point'.
• The set point represents the level or point where a variable physiological state tends to stabilize.
• Hormones regulate the activity of physiological systems.
• Hormone release into the blood is controlled by a stimulus.
• The response to a stimulus changes internal conditions.

Feedback Regulation

• A self-adjusting mechanism by the internal system
• Consists of positive and negative feedback regulation types.

Mechanisms of Regulation

• Osmoregulation
• Thermoregulation
• Chemical Regulation (hormonal)

Positive Feedback Regulation

• Less common in biological systems
• It speeds up the direction of change.
• Lactation (milk production) is an example
  • Suckling stimulates nerve receptors
  • Impulses travel to the pituitary gland
  • This then produces hormones like prolactin and oxytocin
  • These travel in circulation to the mammary gland.
  • This stimulates milk production and ejection.

Negative Feedback Regulation

• Most common feedback loop in the biological system.
• It acts to reverse the direction of change to maintain the set point.
• An increase in carbon dioxide (CO2) level in the air is an example
  • The lungs are signaled to exhale more carbon dioxide.
  • An increased breathing rate results.
  • This causes balance of CO2 levels in the lungs and the amount of oxygen available for gaseous exchange.
  • This is all for thermoregulation

Osmoregulation

• Involves the movement of water between circulation, cellular compartments, and interstitial space.
• It ensures a balance of water and solutes across cell membranes.
• Maintaining this provides optimal functioning of biochemical processes.
• Two conditions alter the biochemical process, resulting in cell death:
  • An increase in solutes above normal in the extracellular fluid where intracellular fluid moves to the extracellular surface, causing cell shrinkage.
  • A decrease in solutes in the extracellular fluid, causing it to move into cells, leading to cell swelling and potential rupture.
• Hyponatremia occurs when the solution outside of the cell has a lower concentration than the inside of the cell; water will move into the cell by osmosis, sometimes causing it to burst.
• Hypernatremia the solution outside of the cell is more concentrated than the inside of the cell; water will move out of the cell by osmosis, causing it to shrink.

Enzymes

• Protein Catalysts
• List the characteristics and functions of enzymes.

Enzyme Characteristics

• Protein catalysts that accelerate chemical reactions.
• Mediators of biochemical reactions.
• They remain unchanged in the overall process.
• Enzymes selectively channel substrates into useful pathways.
• Enzymes to some extent - directs all metabolic events

Nomenclature for Enzymes

• Recommended names typically include:
  • The suffix "-ase" attached to the substrate of the reaction, as in glucosidase, or
  • A description of the action performed, as in lactate dehydrogenase.
• Systematic names
  • Names are divided into six major classes, with subgroups within each class.
  • The suffix -ase is attached to a description of the chemical reaction catalyzed.
  • It usually includes the names of all the substrates, such as lactate: NAD+ oxidoreductase.

Six Classes of Enzymes

• Oxidoreductases catalyze oxidation-reduction reactions.
• Transferases catalyze the transfer of chemical groups.
• Hydrolases catalyze the cleavage of bonds by adding water.
• Lyases catalyze the cleavage of C-C, C-S, as well as certain C-N bonds.
• Isomerases catalyze racemization of optical or geometric isomers.
• Ligases catalyze the formation of bonds between carbon, O, S, and N.
  • Formation is coupled to the hydrolysis of high-energy phosphates.

Enzyme Function

• Enzymes contain a special pocket or cleft called the active site.
• Active sites contain amino acid side chains.
  • These participate in substrate binding and catalysis.
• When the substrate binds to the enzyme, this forms an enzyme-substrate (ES) complex.
• Binding causes a conformational change in the enzyme, also known as induced fit, that allows catalysis.
• The ES is converted into an enzyme-product (EP) complex.
  • This subsequently dissociates into enzyme and product.

Catalytic Strategies

• The type of strategy is chosen based on structural properties and the reaction that the enzyme will catalyze.
• A combination of strategies can be used in catalyzing reactions.
• Includes Covalent Catalysis, Acid-Base Reactions, Catalysis by Approximation, and Metal Ion Catalysis.

Covalent Catalysis

• In covalent catalysis, enzymes or cofactors covalently bond to the substrate as the first step.
• Active sites contain a reactive group that forms a covalent bond with the substrate.
• The enzyme undergoes a mechanism, breaking down the substrate and reforming itself.
• Example: Chymotrypsin uses a serine reside as a nucleophile to attract an unreactive carbonyl group of a substrate.

Acid-Base Reactions

• Enzymes use a molecule other than water to donate or accept protons as a nucleophile.
• Zinc ions complexed with histidine in carbonic anhydrase break down H2CO4 into hydrogen ions and bicarbonate ions.
• Zinc attracts a water molecule, which then deprotonates.
• Oxygen here acts as a nucleophile and attacks a carbon dioxide molecule.
  • This creates a complicated coordination complex

Catalysis by Approximation

• Closeness of two substrates can accelerate the reaction rate between the two.
• Entropy generally decreases when the reaction occurs.
• An increase in reactant concentration occurs with an enzyme bringing two molecules together.

Metal Ion Catalysis

• Metal ions directly facilitate forming bond.
• They can be electrophilic to stabilize the charges on the intermediates of the rxn.

Enzyme properties

• Active Sites
• Catalytic Efficiency
• Specificity
• Holoenzymes
• Regulation
• Location

Catalytic Efficiency

• Enzyme-catalyzed reactions are very efficient, ~ 10^3-10^8 times faster than uncatalyzed reactions.
• Turnover number measures the number of substrate molecules converted to product per enzyme molecule per second. It is called kcat, and is typically 102-104 s-1

Specificity

• Enzymes interact with one or a few substrates. They catalyze only one type of chemical reaction.
• The set of enzymes made in a cell determines which metabolic pathways occur in that cell.

Holoenzymes

• Some enzymes require molecules other than proteins for enzymatic activity.
  • Holoenzyme: an active enzyme with its non-protein component (cofactor)
  • Apoenzyme: inactive enzyme without its nonprotein moiety (cofactor)
• Nonprotein moieties include:
  • Metal ions (e.g., Zn2+ or Fe2+), which act as cofactors.
  • Small organic molecules, which act as coenzymes.
• Cofactors non-protein helper molecules required for apoenzymes or enzymes made of conjugated proteins.
• Coenzymes inactive non-protein organic co-substrates that participate in catalysis.

Location and Regulation of Enzymes

• Enzymes are localized in specific organelles within the cell.
  • Compartmentalization isolates reaction substrates or products from competing reactions.
  • Localising to organelles provides a favorable and organized environment for reactions
• Enzyme activity is regulated—increased or decreased—so the product formation responds to cellular need.

Mechanism of Enzyme Action

• Two main perspectives on the mechanism:
  • Catalysis is viewed in terms of energy changes during a reaction; Enzymes facilitate an alternate, energetically favorable reaction differing from an uncatalyzed one.
  • This describes how the active site chemically simplifies catalysis.
• The first treats catalysis in energy change terms during rxn, enzymes provide for alternation, energetically favorable rxn alternative. ∆G = Gibbs free energy

The Second Perspective

• Also known as the induced fit model, the active site chemically facilitates catalysis.

Factors that affect enzyme function

• Substrate concentration: Includes maximal velocity and hyperbolic shape of the enzyme kinetics curve.
  • Higher concentrations of substrate create sigmoidal curve of velocity
• Temperature: Increase of velocity with temperature, and then a decrease of velocity with higher temperature
• pH: Related to Ionization of active site, Denaturation, and Varying optimum pH.

Michaelis-Menten Reaction Model

k1 k2

• E + S ES E + P k-1
• E is enzyme, S is substance, ES is enzyme-substrate complex
• P is product; and k1, k-1,& k2 are rate constants.

Michaelis-Menten Equation

• Michaelis-Menten Equation describes how reaction velocity varies with concentration of substrates vo = (Vmax [S]) / (Km + [S])
• vo = initial reaction velocity
  • Vmax = maximal velocity
  • Km = Michaelis constant = (k-1 + k2)/k1 -[S] = substrate concentration
• Based on two assumptions: Relative [E] & [S] and Steady-state

Characteristics of Km

• The characteristic of an enzyme and its particular substrate reflects the enzyme's affinity for that substrate.
• Km equals the substrate concentrated at which the rxn velocity is equal to 1 /2Vmax

Enzyme Inhibition

• A substance that diminishes the velocity of an enzyme-catalyzed reaction is called an inhibitor.
• Irreversible inhibitors bind through covalent bonds.
• Reversible inhibitors bind through noncovalent bonds.
• Two types of inhibition:
  • Competitive
  • Non-competitive

Competitive Inhibition

• The inhibitor binds reversibly the the substrate.
• As a result, competes with the substrate for the enzyme's active binding site.

Non-Competitive Inhibition

• The inhibitor and substrate bind at different sites on the enzyme.
• Can bind the free enzymes or the ES complex

Enzyme Regulation

• Is essential to coordinate its numerous metabolic processes.
• Enzymes are very responsive to changes in substrate concentration
• Enzymes respond through increased/decreased rxn rate.
• Enzymes with specialized regulatory functions respond to allosteric effectors or covalent effectors.

Allosteric Regulation of Enzyme Activity

• Allosteric are regulatd by effectors molecules
• Usually composed of multiple subunits
• The regulatory (allosteric) site that binds the effector is a subunit that is not catalytic.
• Allosteric effectors alter the affinity of the enzyme for its substrate or modify the catalytic activity of the enzyme.
• Positive effectors increase, vise versa

Covalent modification

• Catalysis includes the:
  • Addition or removal of phosphate groups from specific serine, threonine, or tyrosine residues of the enzyme.
• Protein phosphorylation is a primary way in which cellular processes are regulated.
• Phosphorylation and dephosphorylation:
  • Kinases catalyze phosphorylation reactions using adenosine triphosphate (ATP) as a phosphate donor.
  • Phosphoprotein phosphatases cleave phosphate groups from phosphorylated enzymes.

Enzyme Regulation

• Regulation of enzyme activity varies based on event and regulation type

Carbohydrate Metabolism

• Related to glucose uptake

Glucose Uptake

• Insulin binds to receptor and the signal cascade mobilizes GLUT.
  • GLUT brings in Glucose and phosphorylates by consumption in energy by Hexokinase

GLUT: Glucose Transporters

• GLUT I: Found in blood (RBCs), baby (foetus), and BBB (blood-brain barrier)
• GLUT II: Found in Ki (kidney), Li (liver), and Ps (pancreas)
• GLUT III: Found in P (placenta), N (neurons), and K (kidney)
• GLUT IV (insulin dependent): Found in M (muscles) and F (fat cells -adipocytes)

Glycolysis

• List substrates, important intermediates, and products of glycolysis
• Describe the conditions that determine the route of pyruvate
• Tabulate regulatory enzymes of glycolysis along with factors that regulate these enzymes.