Biochemistry Protein Structure and Function

Building Blocks of Proteins: Amino Acids

• Proteins are polymers of amino acids linked by peptide bonds.
• There are 20 common amino acids used to build proteins.
• All amino acids have an α-amino group, an α-carboxyl group, and an R-group (side chain) attached to a central α-carbon.
• The R-group dictates the amino acid's properties and influences the protein's overall structure and function.
• Amino acids are classified based on the chemical properties of their R-groups: non-polar, polar, acidic, basic, or aromatic.
• Some amino acids have ionisable groups in their R-groups, contributing to the net charge of the protein.
• The isoelectric point (pI) of a protein is the pH at which the net charge of the protein is zero.
• Peptide bonds are formed between the α-carboxyl group of one amino acid and the α-amino group of another.
• The peptide bond has partial double bond character, making it planar.
• The peptide bond is usually in the trans configuration, with the R-groups on opposite sides of the peptide bond.
• The primary structure of a protein is the linear sequence of amino acids linked by peptide bonds.
• The amino acid with the unlinked α-amino group is called the N-terminus, and the amino acid with the unlinked α-carboxyl group is called the C-terminus.
• Amino acid sequences are written from N-terminus to C-terminus.
• The primary sequence determines the protein's higher-order structure and ultimately its function.

Post-Translational Modifications

• Proteins undergo modifications after translation, termed post-translational modifications.
• These modifications include proteolytic cleavage, glycosylation, lipid addition, disulfide bridge formation, hydroxylation, C-terminal amidation, and phosphorylation.
• These modifications are crucial for generating the correct active form of the protein.
• Proteins also undergo age-related modifications like glycation and free radical damage.
• Protein function is influenced by its amino acid sequence, higher order structure, and post-translational modifications.

Levels of Protein Structure

• The primary structure refers to the amino acid sequence linked by peptide bonds.
• The secondary structure involves local regular folding stabilized by hydrogen bonds between backbone peptide groups, forming structures like α-helix, β-sheet, and turns.
• The tertiary structure refers to the three-dimensional arrangement of the complete polypeptide chain.
• The quaternary structure describes the association of two or more polypeptide chains in a multi-subunit protein.
• Primary structure determines all higher levels of structure, and the 3-dimensional structure of a protein dictates its biological function.

Secondary Structure

• Secondary structures arise from favored rotations of peptide backbone bonds.
• The two most stable secondary structures are the α-helix and β-sheet (parallel and antiparallel).
• The α-helix has 3.6 residues per turn and a pitch of 5.4 Å, with hydrogen bonds forming between carbonyl oxygen and amide -NH groups.
• The β-sheet consists of extended peptide backbones with hydrogen bonds between different parts of the polypeptide chain.
• The presence of certain amino acids like proline and glycine can hinder α-helix formation, and sidechain size and charge influence β-sheet structure.

Other Secondary Structures

• β-turns allow polypeptide chains to change direction and are often found between strands of anti-parallel β-sheets.
• Proline is commonly found in β-turns due to its natural bend.

Tertiary Structure

• Tertiary structure refers to the 3-dimensional arrangement of a protein, stabilized by long-range interactions between amino acid residues.
• Tertiary structure is maintained by non-covalent interactions and disulfide bonds in extracellular proteins.
• Hydrophobic residues cluster inside the protein, while polar residues are on the outside.
• Hydrogen bonds, ionic bonds, and metal ions contribute to protein stability.

Protein Domains

• A domain is a stable, independently folded region within a protein's tertiary structure.
• Proteins can contain one or more domains, often associated with specific functions.
• Different protein families share similar structures despite variations in primary sequences.

Quaternary Structure

• Quaternary structure involves non-covalent interactions between two or more folded polypeptide chains (subunits).
• These assemblies are named based on the number of subunits, e.g., dimers, trimers, tetramers.

Protein Folding

• Protein folding is driven by hydrophobic interactions, with internal hydrophobic residues directing folding to the native conformation.
• Formation of secondary structures (α-helix and β-sheet) initiates folding, followed by the assembly of these structures into larger units.
• Protein folding is an ordered and cooperative process, with each folding step facilitating the next.

Denaturation and Renaturation

• Denaturation disrupts the native structure of a protein, causing loss of function.
• Denaturation can be induced by heat, detergents, solvents, and extremes of pH.
• Some denaturation can be reversed, depending on the severity and the protein.
• Anfinsen's experiment demonstrated that the primary sequence contains all the information necessary for determining the 3-dimensional structure.

Molecular Chaperones

• Molecular chaperones assist protein folding by preventing improper folding.
• Chaperonins are large multi-subunit proteins that provide an environment for protein folding.

Misfolded Proteins and Disease

• Misfolding and aggregation of proteins contribute to diseases like prion diseases, Alzheimer's Disease, and Type II diabetes.
• Amyloid is a misfolded protein believed to play a role in pathogenesis.

Prion Diseases

• Prion diseases are infectious diseases caused by prion proteins, which are abnormally folded versions of normal cellular proteins.
• The pathogenic form (PrPSc) leads to the formation of stable aggregates, resistant to proteases, forming plaques in infected cells.
• PrPSc can convert the normal form of the protein to the abnormal conformation.
• Prion diseases have been identified in cows, sheep, and humans.

Enzymes as Biological Catalysts

• Enzymes are biological catalysts that accelerate reactions without altering reaction equilibrium.
• They achieve this by lowering the free energy of activation, making it easier to reach the transition state.

Features of Enzyme-Catalyzed Reactions

• Enzymes significantly increase reaction rates compared to uncatalyzed or chemically catalyzed reactions.
• Enzyme-catalyzed reactions proceed under mild conditions (temperature and pH) found in cells.
• Enzymes exhibit high substrate specificity, crucial in complex intracellular environments.
• Enzyme activity is tightly regulated, ensuring efficient function.

Enzyme Nomenclature

• Enzyme names often reflect the type of reaction they catalyze, for example, adding "-ase" to the substrate name.
• The International Enzyme Commission has standardized enzyme classification, categorizing enzymes based on their reaction types.

Enzyme Classification and Nomenclature

• Enzymes are classified based on the type of reaction they catalyze, the group donors, and the group acceptors.
• They are assigned code numbers according to these classifications.
• Many enzymes require cofactors, which are small, inorganic molecules often derived from minerals.

Catalytic Mechanisms

• Enzymes employ multiple catalytic mechanisms to facilitate chemical reactions, including covalent catalysis, acid-base catalysis, redox reactions, and the use of cofactors.
• Covalent Catalysis involves the transient formation of covalent bonds between the substrate and an active site residue, often with a cofactor.
• Acid-Base Catalysis involves the donation or acceptance of a proton by acidic or basic groups within the enzyme, common residues include Glu, Asp, Lys, Arg, His, and Cys.
• Metal Ion Cofactors can play various roles in the catalytic process, including nucleophile or electrophile formation, substrate binding, and charge stabilization.

Enzyme-Substrate Interactions

• Enzymes bind substrates at active sites through specific interactions.
• Active sites are three-dimensional regions with a specific shape and amino acid composition.
• Lock and Key Model: Enzyme and substrate are perfectly complementary in shape.
• Induced Fit Model: Binding of substrate induces conformational changes in the enzyme, optimizing the fit.

Enzyme Kinetics

• Enzymes accelerate reactions by lowering the activation energy (ΔG‡) without altering the overall free energy change (ΔG).
• Enzymes can influence the rate of a reaction through various mechanisms, including:
  • Ground State Destabilization: Binding to the active site increases substrate energy.
  • Transition State Stabilization: Enzymes stabilize the high-energy transition state, lowering its free energy.

Measuring Enzyme Activity

• Enzyme activity is measured by determining the initial rate (velocity) of the reaction (V).
• The initial rate is defined as the rate of product formation at time zero.
• Michaelis-Menten Kinetics describes the relationship between substrate concentration ([S]) and reaction velocity (V):
  • Vmax: The maximum reaction velocity achieved at high substrate concentrations.
  • KM: The Michaelis constant, represents the substrate concentration at which the reaction proceeds at half its maximum velocity.
• Kcat: The turnover number, measures the number of substrate molecules converted to product per enzyme molecule per unit time.
• Kcat/KM: A measure of catalytic efficiency, reflecting the enzyme's ability to bind and convert substrate.

Transition State Analogs as Drugs

• Transition State Analogs are molecules that mimic the structure of the transition state.
• They bind tightly to enzyme active sites and inhibit enzyme activity.
• They are potential targets for drug development.

Michaelis-Menten Equation

• Can be rewritten as a linear equation
• Plotting 1/V against 1/[S] will give straight line
• KM is determined from intercept on X-axis
• Vmax is determined from the intercept on Y-axis

Michaelis-Menten Constant (Km)

• Measures the substrate concentration at ½ Vmax
• Provides a measure of how tightly substrate is bound to enzyme
• Smaller KM value, the tighter the substrate is bound

Maximal Velocity (Vmax)

• Represents the maximum rate of reaction
• Can be determined from the enzyme's turnover number
• Turnover number also represented as kcat
• Kcat is calculated by Vmax/[Et] where [Et] is total enzyme concentration

Enzyme Efficiency

• Measured by the ratio kcat/KM
• Represents the speed of product formation from binding substrate
• A higher ratio indicates more efficient enzyme

Enzyme Inhibition

• Enzyme inhibitors prevent normal enzyme function
• Reversible inhibitors can be competitive or non-competitive
• Irreversible inhibitors bind covalently with enzyme and deactivate it permanently

Competitive Inhibition

• Inhibitor molecule competes with substrate at active site
• Inhibitor binding prevents the substrate from binding
• Can be overcome by increasing substrate concentration
• Increased KM but Vmax remains the same

Non-competitive Inhibition

• Inhibitor binds at a site on the enzyme other than the active site
• Binding can alter enzyme activity and/or substrate binding
• Pure non-competitive inhibition does not affect KM
• Mixed non-competitive inhibition increases KM

Allosteric Enzymes

• Exhibit a sigmoidal curve of V vs [S] due to cooperative subunit interaction
• Multiple protein subunits
• Contain an allosteric bind (different from the active site) where modulator molecules bind
• Modulators can be activators or inhibitors

Activator

• Stabilize the R state which has a high affinity for substrate
• V vs [S] curve becomes more hyperbolic and shifts to the left

Inhibitor

• Stabilize the T state which has a low affinity for substrate
• V vs [S] curve becomes more sigmoidal and shifts to the right

Haemoglobin

• Primary protein inside red blood cells
• Responsible for oxygen transport in the bloodstream
• Contains both globin and haem
• Iron in haem is vital to oxygen binding, and exists as Fe2+
• Haemoglobin exists in two states: R-state (oxyhaemoglobin, oxygen bound) and T-state (deoxyhaemoglobin, oxygen unbound)

Myoglobin

• Related to haemoglobin, but found in muscle cells
• Acts as an oxygen reservoir for muscle activity
• Contains a single globin and haem unit, is a monomer
• Has a higher oxygen affinity than haemoglobin, leading to tight oxygen binding

Globin Fold

• Characteristic shape of globin proteins
• Consists of 8 α-helical regions labelled A-B-C-D-E-F-G-H
• Fold is similar across different globin types

Oxygen Binding Properties

• Haemoglobin has a sigmoidal oxygen binding curve, exhibiting cooperativity
• Myoglobin displays a hyperbolic curve
• Myoglobin binds oxygen more tightly than haemoglobin
• Haemoglobin releases oxygen at lower oxygen pressures in tissues

Haem

• Consists of a porphyrin ring with four pyrrole rings
• Contains ferrous iron (Fe2+) at the centre, which is oxygenated
• Haem-globin combination prevents oxidation of Fe2+, enabling reversible oxygen binding

Cooperativity

• Occurs in haemoglobin due to its tetrameric structure
• Leads to conformational changes between the R and T states
• R-state has high oxygen affinity
• T-state has low oxygen affinity
• All four globins within haemoglobin are in the same state (either R or T)

Allosteric Regulation

• Modulator molecules bind at allosteric sites, altering enzyme activity
• Can be activators or inhibitors
• Activators promote substrate binding and shift V vs [S] curve to left
• Inhibitors suppress substrate binding and shift V vs [S] curve to right

Multienzyme Metabolic Pathways

• Chains of enzymes where product of one reaction becomes substrate for the next
• Allosteric enzymes play a crucial role in regulating these pathways
• Rate limiting enzymes control the overall pathway speed

Glycogen Phosphorylase

• Key enzyme in glycogen breakdown (glycogenolysis)
• Allosterically regulated by both activators and inhibitors
• AMP acts as an activator, promoting glycogen breakdown
• Glucose-6-phosphate acts as an inhibitor, preventing glycogen breakdown

Haemoglobin Structure and Function

• Haemoglobin is a protein responsible for oxygen transport in the blood.
• It consists of four subunits: two alpha and two beta globin chains.
• Each subunit contains a heme group, which binds to oxygen.
• Oxygen binding to one heme group triggers a conformational change that makes it easier for other heme groups to bind oxygen, increasing oxygen uptake.
• This process is called cooperativity.
• Deoxyhaemoglobin (deoxygenated) has a dished haem, while in oxyhaemoglobin (oxygenated), oxygen flattens the haem and pulls histidine F8 and helix F toward the binding site.

Allosteric Regulation of Haemoglobin

• 2,3-Bisphosphoglycerate (BPG) is an allosteric regulator of haemoglobin.
• BPG binds to a positively charged pocket in the center of the haemoglobin tetramer, stabilizing the T state (low-affinity state).
• BPG binding prevents haemoglobin from readily switching to the R state (high-affinity state), thus reducing oxygen binding affinity.
• BPG is produced when glucose is broken down for energy production.
• In the lungs, high oxygen concentration pushes BPG off haemoglobin, favouring the oxygenated R state.
• Other allosteric inhibitors, like CO2 and H+, also stabilize the T state, increasing cooperativity and promoting oxygen release in tissues.

Haemoglobin Variants

• Foetal haemoglobin has a higher affinity for oxygen than adult haemoglobin, facilitating oxygen transfer from mother to foetus.
• Foetal haemoglobin contains gamma subunits instead of beta subunits.
• The gamma subunit has a serine at position 143, while the beta subunit has a histidine at that position.
• This difference in amino acid composition results in a weaker binding of BPG to foetal haemoglobin, favoring the R state and higher oxygen affinity.

Sickle Cell Haemoglobin

• Sickle cell anaemia results from a point mutation in the beta globin chain, replacing glutamate with valine at position 6.
• This mutation causes haemoglobin S to polymerize when deoxygenated, distorting red blood cells into a sickle shape.
• Sickled red blood cells are less flexible and can block capillaries, leading to various health complications.

Receptor Classes

• Receptors are proteins that bind to ligands and initiate cellular response.
• Four main types of receptors:
  • Ligand-gated ion channels
  • G-protein coupled receptors (GPCRs)
  • Receptor tyrosine kinases (RTKs)
  • Nuclear receptors
• Ligand-gated ion channels open upon ligand binding, allowing ion flow across the membrane and causing a rapid cellular response.
• GPCRs activate G proteins, which then initiate a signal transduction cascade using second messengers like cyclic AMP (cAMP), leading to a slower but more complex cellular response.
• RTKs phosphorylate adaptor proteins upon ligand binding, initiating a signaling cascade and ultimately leading to cellular responses.
• Nuclear receptors regulate gene expression by binding to specific DNA sequences in the nucleus.

Signal Transduction

• Signal transduction is a process where a signal is passed from the membrane into the cell, eliciting a cellular response.
• The signal transduction pathway is unique for each receptor class.
• Ligand-gated ion channels generate a rapid response by directly altering membrane potential.
• GPCRs utilize a complex cascade of events involving second messengers and phosphorylation, leading to a slower but more diverse cellular response.
• RTKs initiate a signaling cascade through phosphorylation of adaptor proteins, ultimately leading to cellular responses.

Enzymes vs. Receptors

• Both enzymes and receptors bind molecules and undergo activation and inhibition.
• Enzymes transform their substrates into products, while receptors activate or inhibit cellular responses without altering the bound ligand.
• Enzymes generally have one active site, while receptors may have multiple ligand binding sites.