Metabolism 1: An Introduction to Protein Structure

Outline the reaction by which amino acids are joined together; sketch a trimeric peptide, illustrating the amino terminus, carboxyl terminus and side chains

Protein: any of a group of organic compounds composed of one or more chains of amino acids and forming an essential part of all living organisms

  • Human body: 20% protein
  • Protein mutations are responsible for a variety of diseases (e.g. sickle cell anaemia)

Structure of an amino acid:

  • Substitutions of the side chain(R group) give rise to the 20 different amino acids
  • Backbone: the whole of the amino acid minus the side chain

Amino acids with hydrophobic side chains / Amino acids with hydrophilic side chains
Glycine: the simplest amino acid (R group = H) / Asparagine
Alanine / Glutamine
Valine / Cysteine
Leucine / Histidine
Isoleucine / Serine
Proline / Threonine
Methionine / Tyrosine
Phenylalanine / Tryptophan
Aspartate
Glutamate
Lysine
Arginine
Amino acids with charged side chains
Arginine and Lysine / Protonated at physiological pH; therefore basic
Histidine / Often protonated
Aspartic acid and Glutamic acid / Deprotonated at physiological pH; therefore acidic

Amino acids with charged side chains:

  • The ionisation state of an amino acid provides vital biological properties to many proteins
  • Therefore cells cannot generally tolerate wide changes in pH
  • If the ionisation state of key amino acids within a protein is altered: a loss of biological activity often results
  • The ability to take up and release protons gives amino acids some buffering capacity to resist some changes in pH

Chirality:

  • The central Cαcarbon atom is a chiral centre: i.e. it has 4 different substituents bound to it
  • This produces optical isomers (enantiomers) of each amino acid, which are non-superimposable mirror images of each other
  • Glycine (Gly) has no side chain; therefore it is the only non-chiral amino acid
  • All amino acids found in proteins are L-enantiomers

Peptides are formed by a condensation reaction between 2 amino acids:

Structure of a peptide:

Protein structure:

  • The polypeptide chain of a protein rarely forms a disordered structure
  • Proteins have functions which rely upon specificity
  • Functionality requires a definite 3D structure (conformation) of the polypeptide chain
  • Proteins generally possess a degree of flexibility which is necessary for function (e.g. muscle fibres)

Characteristics of the peptide bond:

  • There is no free rotation about the peptide bond
  • The C=O and N–H groups are in the same plane of the molecule
  • The other 2 bonds in the backbone of the polypeptide chain are able to rotate

Understand the concepts of primary structure, secondary structure, tertiary structure & quaternary structure with respect to proteins

Folding of proteins:

  • Proteins generally fold into a single conformation of lowest energy
  • Folding of proteins may occur spontaneously or it may involve chaperones

Chaperones: molecules which bind to a partially folded polypeptide chain and ensure that folding continues along the most energetically favourable pathway

  • By breaking the bonds which hold a protein together, a protein is denatured into the original flexible polypeptide

Common laboratory denaturants include:

  • Urea: breaks hydrogen bonds
  • 2-mercaptoethanol: breaks disulphide bonds

Structural levels of proteins:

  • Primary structure: the linear sequence of amino acids which constitute the protein

Nomenclature: the protein sequence is written from the amino terminus to the carboxyl terminus

  • Secondary structure: local structural motifs within a protein (e.g. α-helices and ß-pleated sheets)
  • Tertiary structure: arrangement of motifs of the secondary structure into domains (compact globular structures)
  • Quaternary structure: the 3D structure of a multimeric protein, composed of several subunits

Distinguish between anα-helix and a ß-pleated sheet and appreciate the bonds that stabilise their formation

α-helices:

  • Hydrogen bonds between the C=O group of 1 residue and the N–H group of another residue stabilise the helix
  • Side chains of individual amino acids project out from within the helix
  • Right-handed helices are more common since L-enantiomers of amino acids are used in proteins
  • Proline: when proline is joined to a polypeptide chain, it loses its N–H group

This prevents the N atom from hydrogen bonding with the C=O group of another residue within the helix

The helical conformation is distorted and kinked

ß-pleated sheets:

  • Hydrogen bonds between the C=O and N–H groups of 2 or more ß-strands stabilise the sheet
  • C=O and N–H groups project out perpendicular to the line of the backbone
  • Parallel ß-pleated sheet: alternate ß-strands run in the same direction
  • Anti-parallel ß-pleated sheet: alternate ß-strands run in opposite directions
  • Pleating allows for the best alignment of the hydrogen-bonded C=O and N–H groups

Appreciate the different types of bond that combine to stabilise a particular protein conformation

Covalent bonds: 2 atoms share electrons

  • The strongest bonds within a protein
  • Exist in the primary structure
  • May exist as disulphide bridges:

Disulphide bridges occur when cysteine side chains within a protein are oxidised

This results in a covalent link between the 2 amino acids

–CH2-SH + HS-CH2––CH2-S-S-CH2–

Hydrogen bonds: 2 atoms with partial negative charges share a partially positively charged hydrogen atom

  • May occur either between atoms on different Side chains and the backbone of the protein or between water molecules

Ionic interactions: electrostatic attractions between charged side chains

  • Relatively strong bonds, particularly when the Side chains are within the interior of the protein and excluded from water
  • The majority of charged Side chainsare at the surface of a folded protein, where they can be neutralised by counter-ions (e.g. salts)

Van der Waals forces: electrostatic attractions between 2 atoms due to the fluctuating electron cloud surrounding each atom, which has a temporary electric dipole

  • Weak and transient forces
  • Due to the sheer number of Van der Waals interactions within a protein, they play a large part in the overall conformation of a protein
  • Van der Waals radius: the appropriate distance required for Van der Waals attractions

This varies for different atoms, based on the size of the electron cloud

  • If the 2 electron clouds of adjacent atoms are quite close: the transient dipole in 1 atom can induce a complementary dipole in another atom, with weak attractive properties
  • If the 2 electron clouds of adjacent atoms are too close: there are repulsive forces between the 2 atoms due to the electrons

Hydrophobic interactions:

  • Pack hydrophobic side chains into the interior of the protein
  • This creates a hydrophobic core and a hydrophilic surface

Give examples of the post translational modifications of amino acids, with reference to glycosylation, hydroxylation and carboxylation

Amino acids may be modified following protein synthesis

Hydroxylation:

  • Proline  hydroxyproline

This requires: prolyl hydroxylase and vitamin C

  • Hydroxyproline is present in collagen fibres: additional hydroxyl groups help to stabilise the fibres
  • Scurvy: caused by vitamin C deficiency

Glycosylation:

  • N-linked glycosylation of asparagine: addition of sugar residues
  • This increases the solubility of proteins and protects them from enzymatic degradation
  • Primary structure motif: N-X-S/T (asparagine-any amino acid-serine/threonine)
  • Carbohydrate-deficient glycoprotein (CDG): associated with N-linked sugar chain transfer deficiency

Carboxylation:

  • Glutamate γ-carboxyglutamate

This requires vitamin K-dependent carboxylase

  • Formation of γ-carboxyglutamate residues within several proteins of the blood clotting cascade is critical for their normal function: it increases their calcium binding capability
  • Warfarin(anticoagulant): inhibits the above carboxylation reaction

Lecture summary:

  • Proteins are chains of amino acids linked by peptide bonds which have evolved to fulfil specific functions within the cell
  • Such functions rely upon the conformation (3D structure) of the protein which is determined by a variety of forces
  • The α-helix and ß-pleated sheet are the two staple motifs that define the conformation of a protein
  • The nature of the amino acid side chain dictates its position within the conformation of the protein
  • Post-translational modifications of proteins add more diversity to protein structure

Metabolism 2: Energetics and Enzymes

Define the 1st and 2nd Laws of thermodynamics

1st Law of Thermodynamics: energy can neither be created nor destroyed; it is simply converted from one form to another

2nd Law of Thermodynamics: in any isolated system, such as a single cell or the universe, the degree of disorder can only increase

  • Entropy: the amount of disorder in a particular system
  • Reactions proceed spontaneously towards products with greater entropy
  • Biological systems are very well ordered: energy is taken from the environment and invested into chemical reactions within the cell which maintain order

Explain the concept of free energy and how we can use changes in free energy to predict the outcome of a reaction

Free energy (G) [kJ/mol]: the amount of energy within a molecule that can perform useful work at a constant temperature

∆G: the amount of disorder that results from a particular reaction

For the reaction: A + B  C + D

∆G = G (C + D) − G(A + B)

  • A reaction can only occur spontaneously if ∆G is negative (energetically favourable reactions)
  • A reaction cannot occur spontaneously if ∆G is positive (energetically unfavourable reactions)

Draw the chemical structure of ATP and explain how it acts as a carrier of free energy and is used to couple energetically unfavourable reactions

ATP:

ATP structure:

  • Phosphoanhydride bonds have a large negative ∆G of hydrolysis
  • ATP  ADP + Pi∆G = −31 kJ/mol

Coupled reactions:

  • Biosynthetic pathways are generally energetically unfavourable (e.g. peptide synthesis)
  • They take place because they are coupled to an energetically favourable reaction
  • A reaction will proceed if the sum of ∆G for the overall reaction is positive
  • Most energetically unfavourable biochemical reactions are coupled to the hydrolysis of high-energy phosphoanhydride bonds (e.g. ATP hydrolysis)

Example 1: glucose + fructose  sucrose ∆G = +23 kJ/mol

This reaction is coupled to ATP hydrolysis:

Glucose + ATP  glucose-1-phosphate + ADP

Glucose-1-phosphate + fructose  sucrose

∑∆G = −31 + (+23) = −8 kJ/mol; therefore it is energetically favourable

Example 2: glucose-6-phosphate + H2O  glucose + Pi∆G = −13.8 kJ/mol

This energetically favourable reaction will not occur at a useful rate unless it is catalysed by an enzyme

N.B. enzymes do not change the value of ∆G

Example 3: glucose + oxygen  carbon dioxide + water∆G = −2872 kJ/mol

This energetically favourable reaction results in an increase in the entropy (disorder) of the universe

It does not occur spontaneously since energy must be supplied to overcome the activation energy barrier

Describe how enzymes act as catalysts of reactions with reference to the reaction catalysed by lysozyme

Enzyme: a protein that acts as a catalyst to induce chemical changes in other substances itself remaining apparently unchanged by the process

Mode of action: enzymes lower the activation energy barriers that impede chemical reactions from taking place

  • 1 or more substrates bind to the enzyme tightly at the active site
  • The enzyme arranges the substrate(s) in such a way that certain bonds are strained
  • Key residues within the enzyme participate in either the making or breaking of bonds by altering the arrangement of electrons within the substrate(s)

This can either take the form of oxidation or reduction reactions

Transition state: the particular conformation of the substrate in which atoms of the molecule are rearranged both geometrically and electronically so that the reaction can proceed

  • Enzymes work by bending substrates in such a way that the bonds to be broken are stressed: the substrate molecule resembles the transition state

Lysozyme: a component of tears and nasal secretions, involved in defence against bacteria

Lysozyme catalyses the hydrolysis of sugar molecules within bacterial cell walls: this results in lysis of bacteria

  • Lysozyme hydrolyses alternating polysaccharide copolymers of N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM)
  • Lysozyme hydrolyses the ß-1,4 glycosidic bond which links C1 of NAM to C4 of NAG

Mechanism of action:

  1. Glu35 protonates the oxygen in the glycosidic bond: this breaks the glycosidic bond
  2. Glu35 deprotonates a water molecule: this forms a hydroxide ion
  3. Asp52 stabilises the positive charge on the transition state
  4. The hydroxide ion attacks the transition state and adds an –OH group
  5. Glu35 and Asp52 are in their original state to continue catalysis

Optimum pH for lysozyme: 5.0

At pH 5.0:

  • Asp52 is deprotonated (–COO−)
  • Glu35 is protonated (–COOH)

This is essential for lysozyme function

Describe how oxidation and reduction involve the transfer of electrons

Oxidation: loss of electrons

Reduction: gain of electrons

  • Since the cellular environment is generally aqueous, often when a molecule gains an electron, it simultaneously gains a proton

Outline the reaction catalysed by glucose-6-phosphatase and explain what clinical symptoms are linked to inherited deficiencies of this enzyme

In the liver: glycogen  glucose-6-phosphate  glucose

Glucose-6-phosphate + H2O  glucose + Pi

This reaction is catalysed by glucose-6-phosphatase

  • Glucose-6-phosphatase is predominantly a liver enzyme
  • It catalyses the above reaction when blood glucose levels are low and releases glucose from the liver into the bloodstream

Glucose-6-phosphatase deficiency (Von Gierke’s disease)

Symptoms:

  • Low blood sugar levels
  • Slow growth
  • Large livers
  • Short stature

Outline the differences between lock and key and induced fit models of substrate-enzyme interactions

Lock and key model: the shape of the substrate is complementary to that of the active site of the enzyme

  • This model explains the specificity of most enzymes for a single substrate

Induced fit model: the substrate induces a change in the conformation of the enzyme which results in the formation of the active site; upon release of the products the enzyme reverts back to its original conformation

  • This is the correct model since proteins generally possess a degree of flexibility necessary for function
  • Crystallographic analysis of enzymes supports this model

Describe graphically, the effects of a) substrate concentration, b) temperature and
c) pH on enzyme catalysed reactions

Effect of substrate concentration:

Enzyme kinetics:

Michaelis constant (KM): the concentration of substrate at which a given enzyme works at half its maximal velocity (vmax)

KM is useful to compare the strength of enzyme-substrate complexes:

  • Low KM value: tight binding within the enzyme-substrate complex
  • High KM value: weak binding within the enzyme-substrate complex

At vmax the rate of product formation depends on the turnover number (i.e. how rapidly the substrate can be processed)

Lineweaver-Burk Plot: a double reciprocal plot of 1/V against 1/[S]

Effect of temperature:

Catalysis increases as temperature is increased

Each enzyme has an optimum temperature; above this the enzyme’s conformation is denatured

Effect of pH:

The above graph is typical of most enzymes: they have an optimum pH at which the catalytic side chains are in the correct ionisation state

Illustrate the role of the coenzyme NAD in the reactions catalysed by glyceraldehyde3-phosphate dehydrogenase, lactate dehydrogenase and malate dehydrogenase, referring to the biochemical changes involved in its reduction to NADH

NAD+: a cofactor for dehydrogenation reactions

  • It is a coenzyme which only functions after binding to a protein
  • Dehydrogenases catalyse dehydrogenation reactions:

RCH(OH)R’ + NAD+ RCOR’ + NADH + H+

  • NAD+ catalyses dehydrogenation of substrates by accepting a hydrogen atom and 2 electrons:

NAD+ + H+ + 2e- NADH

Glyceraldehyde 3-phosphate dehydrogenase catalyses…

Glyceraldehyde 3-phosphate + NAD+ + Pi 1,3-bisphosphoglycerate + NADH + H+

  • The substrate is oxidatively phosphorylated

Lactate dehydrogenase catalyses…

Pyruvate + NADH + H+ lactate + NAD+

  • Pyruvate is converted into lactate by anaerobic respiration during intense exercise
  • This generates free NAD+ which is needed by muscle for other reactions
  • Lactate is transported from muscles to the liver by the bloodstream
  • The liver has high NAD+ levels which can be used by lactate dehydrogenase to regenerate pyruvate

Malate dehydrogenase catalyses…

Malate + NAD+ oxaloacetate + NADH + H+

Lecture summary:

  • Energetically unfavourable reactions can occur by coupling them to energetically favourable reactions, generally involving the hydrolysis of high-energy phosphoanhydride bonds
  • Enzymes are specific biological catalysts which increase the rate of biochemical reactions by lowering the activation energy barriers that impede chemical reactions
  • Enzymes are sensitive to extremes of temperature and pH, as they are proteins

Metabolism 3: Metabolic Pathways and ATP Production I

Sketch a cartoon of the three stages of cellular metabolism that convert food to waste products in higher organisms, illustrating the cellular location of each stage

Cellular metabolism: energy is liberated from food molecules to provide energy

3 food molecules are used by cells:

  1. Polysaccharides  simple sugars
  2. Proteins  amino acids
  3. Fats  fatty acids and glycerol

3 stages of metabolism:

  1. Digestion: enzymes liberate small molecules in the small intestines
  2. Cellular metabolism I: small molecules are oxidised to generate ATP and NADH in the cell cytosol
  3. Cellular metabolism II: small molecules are oxidised to generate ATP within the mitochondria

Glucose combustion: single-step reaction

  • The relatively large activation energy is overcome by a heat source
  • Free energy is released as heat

Glucose metabolism: multi-step reaction

  • The relatively small activation energies of each step are overcome by enzymes and body temperature
  • Free energy liberated is invested into carrier molecules, such as ATP
  • This reaction is ~50% efficient

Outline the metabolism of glucose by the process of glycolysis, listing the key reactions, in particular those reactions that consume ATP and those that generate ATP

Glycolysis: an anaerobic process which occurs in the cell cytoplasm

  1. Glucose + ATP  glucose 6-phosphate + ADP + H+

Hexokinase

Glucose is committed to further reactions and it is trapped inside the cell (due to negative charge)

  1. Glucose 6-phosphate  fructose 6-phosphate

Phosphoglucose isomerase

  1. Fructose 6-phosphate + ATP  fructose 1,6-bisphosphate + ADP

Phosphofructokinase

Fructose 1,6-bisphosphate is a highly symmetrical, high energy compound

  1. Fructose 1,6-bisphosphate  glyceraldehyde 3-phosphate + dihydroxyacetone phosphate

Aldolase

  1. Dihydroxyacetone phosphate  glyceraldehyde 3-phosphate

Triose phosphate isomerase

  1. Glyceraldehyde 3-phosphate + NAD+ + Pi 1,3-bisphosphoglycerate + NADH

Glyceraldehyde 3-phophate dehydrogenase

  1. 1,3-bisphosphoglycerate + ADP  3-phosphoglycerate + ATP

Phosphoglycerate kinase