Unit 3: Proteins and Enzymes - Subjective Questions

Proteins are high molecular weight biopolymers made up of amino acids linked together by peptide bonds (). They are the most abundant organic molecules in living cells.

Structural Classification of Proteins:

Based on their shape and structure, proteins are classified into three types:

1. Fibrous Proteins:

   • Have elongated, thread-like structures.
   • Polypeptide chains run parallel to form fibers.
   • Insoluble in water.
   • Provide structural and mechanical support.
   • Examples: Collagen, Keratin, Elastin, Fibroin.

2. Globular Proteins:

   • Have compact, spherical/globular shapes.
   • Polypeptide chains are folded into a rounded shape.
   • Soluble in water and dilute salt solutions.
   • Perform dynamic functions.
   • Examples: Hemoglobin, Enzymes, Albumin, Insulin.

3. Membrane Proteins:

   • Associated with cell membranes.
   • Involved in transport and signaling.
   • Examples: Ion channels, Receptors.

Based on Composition:

• Simple Proteins: Yield only amino acids on hydrolysis (e.g., Albumins).
• Conjugated Proteins: Contain a non-protein prosthetic group (e.g., Hemoglobin, Glycoproteins).
• Derived Proteins: Products of protein degradation (e.g., Peptones).

Proteins perform a wide variety of essential functions in living organisms:

1. Catalytic Function (Enzymes):

   • Enzymes are proteins that catalyze biochemical reactions.
   • Example: Amylase, Pepsin, DNA Polymerase.

2. Structural Function:

   • Provide mechanical support to cells and tissues.
   • Example: Collagen (connective tissue), Keratin (hair, nails).

3. Transport Function:

   • Carry molecules across membranes and in blood.
   • Example: Hemoglobin (oxygen transport), Serum albumin (fatty acid transport).

4. Storage Function:

   • Store amino acids and metal ions.
   • Example: Ferritin (iron storage), Casein (milk protein).

5. Hormonal Function:

   • Act as chemical messengers.
   • Example: Insulin, Glucagon.

6. Defense/Protective Function:

   • Protect against pathogens.
   • Example: Immunoglobulins (antibodies).

7. Contractile/Motility Function:

   • Enable movement and muscle contraction.
   • Example: Actin, Myosin.

8. Regulatory Function:

   • Regulate gene expression and cellular activities.
   • Example: Transcription factors.

9. Receptor Function:

   • Receive and transmit signals.
   • Example: Membrane receptors.

Ramachandran Plot is a graphical representation developed by G.N. Ramachandran that plots the dihedral angles (phi) and (psi) of amino acid residues in a polypeptide chain.

Dihedral Angles:

• Phi (): Angle of rotation around the bond.
• Psi (): Angle of rotation around the bond.
• The peptide bond () has partial double-bond character and is rigid ().

Construction:

• The plot has on the x-axis and on the y-axis, both ranging from to .

Significance:

• Allowed regions: Represent sterically favorable combinations of and angles.
• Disallowed regions: Represent sterically unfavorable combinations due to atomic clashes.

Key features observed:

• Right-handed -helix: , .
• -sheet: , .
• Left-handed -helix: Small allowed region.
• Glycine shows more flexibility (no side chain), occupying more area.
• Proline is restricted due to its ring structure.

Uses:

• Validates protein structures determined by X-ray crystallography.
• Predicts secondary structure conformations.
• Identifies errors in protein models.

Proteins exhibit four levels of structural organization:

1. Primary Structure:

   • The linear sequence of amino acids in a polypeptide chain.
   • Amino acids are joined by peptide bonds.
   • Determines all higher-level structures.
   • Example: Insulin sequence.

2. Secondary Structure:

   • Local folding of the polypeptide backbone stabilized by hydrogen bonds.
   • Main types:
     • -helix: Right-handed coil, H-bonds between C=O and N-H four residues apart.
     • -pleated sheet: Extended strands connected by H-bonds.
   • Example: Keratin (-helix), Silk fibroin (-sheet).

3. Tertiary Structure:

   • Overall 3D folding of the entire polypeptide chain.
   • Stabilized by various interactions:
     • Hydrophobic interactions
     • Hydrogen bonds
     • Ionic bonds (salt bridges)
     • Disulfide bonds ()
   • Example: Myoglobin.

4. Quaternary Structure:

   • Association of two or more polypeptide subunits.
   • Stabilized by non-covalent interactions between subunits.
   • Example: Hemoglobin (4 subunits: ).

The stability of a protein's native conformation depends on several stabilizing forces:

1. Covalent Bonds:

   • Peptide bonds: Backbone of the polypeptide chain.
   • Disulfide bonds (): Covalent linkages between cysteine residues that provide strong stabilization.

2. Hydrogen Bonds:

   • Formed between backbone C=O and N-H groups.
   • Stabilize secondary structures (-helix, -sheet).

3. Hydrophobic Interactions:

   • Most important for stability.
   • Nonpolar side chains cluster in the protein interior, away from water.

4. Ionic Interactions (Salt Bridges):

   • Electrostatic attractions between oppositely charged side chains (e.g., and ).

5. Van der Waals Forces:

   • Weak attractions between closely packed atoms.
   • Collectively contribute significant stability.

Thermodynamic Basis:

• Protein stability is measured by the Gibbs free energy of folding:
  
• The native state is only marginally stable (typically ).

Factors affecting stability:

• Temperature
• pH
• Ionic strength
• Presence of denaturants

Protein Denaturation is the process by which a protein loses its native three-dimensional structure (secondary, tertiary, and quaternary) without breaking the peptide bonds (primary structure remains intact). This results in loss of biological activity.

Denaturing Agents:

1. Physical Agents:

   • Heat: Disrupts hydrogen bonds and hydrophobic interactions (e.g., cooking an egg).
   • Radiation: UV rays damage protein structure.
   • Mechanical agitation: Vigorous shaking (e.g., beating egg whites).
   • High pressure.

2. Chemical Agents:

   • Acids and Bases (pH changes): Disrupt ionic interactions.
   • Organic solvents: Alcohol disrupts hydrophobic interactions.
   • Detergents: SDS disrupts hydrophobic core.
   • Chaotropic agents: Urea and guanidine hydrochloride disrupt H-bonds.
   • Heavy metal ions: , bind to sulfur groups.
   • Reducing agents: -mercaptoethanol breaks disulfide bonds.

Effects of Denaturation:

• Loss of biological/enzymatic activity.
• Decreased solubility (precipitation/coagulation).
• Increased viscosity.
• Increased susceptibility to proteolysis.
• Change in physical properties.

Renaturation:

• Some proteins can refold to native state when denaturant is removed (e.g., Ribonuclease - Anfinsen's experiment).

The International Union of Biochemistry (IUB) classifies enzymes into six major classes based on the type of reaction they catalyze. Each enzyme is assigned an EC (Enzyme Commission) number.

1. Oxidoreductases (EC 1):

   • Catalyze oxidation-reduction reactions (transfer of electrons/hydrogen).
   • Example: Lactate dehydrogenase, Oxidases, Catalase.

2. Transferases (EC 2):

   • Catalyze transfer of functional groups from one molecule to another.
   • Example: Hexokinase (transfers phosphate), Transaminases.

3. Hydrolases (EC 3):

   • Catalyze hydrolysis (bond cleavage using water).
   • Example: Amylase, Lipase, Pepsin, Trypsin.

4. Lyases (EC 4):

   • Catalyze addition or removal of groups to form double bonds (non-hydrolytic).
   • Example: Decarboxylase, Aldolase, Fumarase.

5. Isomerases (EC 5):

   • Catalyze intramolecular rearrangements (isomerization).
   • Example: Phosphoglucose isomerase, Epimerase.

6. Ligases (EC 6):

   • Catalyze joining of two molecules coupled with ATP hydrolysis.
   • Example: DNA ligase, Pyruvate carboxylase.

EC Number Format: EC X.X.X.X, e.g., EC 1.1.1.27 for lactate dehydrogenase.

The Michaelis-Menten equation describes the kinetics of enzyme-catalyzed reactions.

Reaction Scheme:

Where E = enzyme, S = substrate, ES = enzyme-substrate complex, P = product.

Derivation (Steady-State Assumption):

At steady state, rate of formation of ES = rate of breakdown of ES:

Let total enzyme , so :

Defining the Michaelis constant:

Solving for :

The velocity and :

Significance of :

• equals the substrate concentration at which .
• Low = high affinity of enzyme for substrate.
• High = low affinity.
• Characteristic constant for each enzyme-substrate pair.

Significance of :

• Maximum reaction rate when the enzyme is fully saturated with substrate.
• Proportional to enzyme concentration.
• Used to calculate turnover number ().

The Lineweaver-Burk plot (double reciprocal plot) is a linear transformation of the Michaelis-Menten equation used to determine kinetic parameters accurately.

Derivation:

Starting from the Michaelis-Menten equation:

Taking reciprocal of both sides:

Separating terms:

This is in the form (straight line equation).

Plot Details:

• X-axis:
• Y-axis:
• Slope:
• Y-intercept:
• X-intercept:

Advantages:

• Gives a straight line, making it easy to determine and accurately.
• Extrapolation gives (difficult to obtain directly from hyperbolic curve).
• Useful for distinguishing between different types of enzyme inhibition (competitive, non-competitive, uncompetitive).

Disadvantages:

• Points at low dominate and can distort the line due to error magnification at small values.

Enzyme activity is influenced by several factors:

1. Substrate Concentration [S]:

   • At low , rate increases linearly.
   • At high , rate plateaus as enzyme becomes saturated (reaching ).
   • Follows Michaelis-Menten kinetics.

2. Enzyme Concentration [E]:

   • Rate is directly proportional to enzyme concentration (when substrate is in excess).
     

3. Temperature:

   • Rate increases with temperature up to the optimum temperature (usually for human enzymes).
   • Beyond optimum, enzyme denatures and activity drops sharply.
   • (rate doubles per rise up to optimum).

4. pH:

   • Each enzyme has an optimum pH.
   • Extreme pH alters ionization of active site groups and can denature the enzyme.
   • Examples: Pepsin (pH 2), Trypsin (pH 8).

5. Inhibitors:

   • Competitive: Compete with substrate for active site.
   • Non-competitive: Bind at allosteric sites.
   • Uncompetitive: Bind only to the ES complex.

6. Activators:

   • Increase enzyme activity (e.g., metal ions like , ).

7. Coenzymes and Cofactors:

   • Required by many enzymes for activity (e.g., NAD, FAD, vitamins).

8. Product Concentration:

   • Accumulation of product may inhibit the reaction (feedback inhibition).

Effect of Temperature:

• As temperature increases, the kinetic energy of molecules increases, leading to more frequent enzyme-substrate collisions and higher reaction rate.
• Activity increases up to the optimum temperature (typically for human enzymes).
• Beyond the optimum, high temperature causes denaturation (breaking of H-bonds and hydrophobic interactions), rapidly decreasing activity.

Temperature graph: A bell-shaped curve peaking at optimum temperature.

Rate
| /\
| / \
| / \
| / \
|/________
Optimum Temp

• The temperature coefficient :
  

Effect of pH:

• Enzymes have an optimum pH at which activity is maximum.
• pH affects the ionization state of amino acid residues at the active site and of the substrate.
• Extreme pH values disrupt ionic bonds and can denature the enzyme.

pH graph: A bell-shaped curve peaking at optimum pH.

Optimum pH examples:

• Pepsin: pH 1.5–2.0 (acidic)
• Salivary amylase: pH 6.8
• Trypsin: pH 8.0 (alkaline)
• Alkaline phosphatase: pH 9.5

Mechanism of Enzyme Catalysis:

Enzymes accelerate reactions by lowering the activation energy () required for the reaction. They achieve this by forming an enzyme-substrate (ES) complex at the active site.

General Steps:

1. Substrate binds to the active site forming the ES complex.
2. Enzyme lowers activation energy and stabilizes the transition state.
3. Product is formed and released, regenerating the free enzyme.

Models of Enzyme-Substrate Binding:

1. Lock and Key Model (Emil Fischer, 1894):

• The active site has a rigid, complementary shape to the substrate.
• The substrate (key) fits exactly into the active site (lock).
• Explains enzyme specificity.
• Limitation: Cannot explain conformational flexibility.

2. Induced Fit Model (Daniel Koshland, 1958):

• The active site is flexible, not rigid.
• When the substrate binds, the active site changes shape to fit the substrate.
• Binding induces a conformational change that positions catalytic groups correctly.
• More widely accepted; explains catalytic action better.

Catalytic Strategies:

• Acid-base catalysis: Transfer of protons.
• Covalent catalysis: Temporary covalent bond with substrate.
• Metal ion catalysis: Metal ions stabilize charges.
• Proximity and orientation effects: Bringing substrates close together.

Enzyme inhibitors reduce enzyme activity. Reversible inhibitors are classified into three types:

1. Competitive Inhibition:

• Inhibitor resembles the substrate and competes for the active site.
• Binds only to free enzyme (E).
• Can be overcome by increasing substrate concentration.
• Effect on kinetics:
  • increases (apparent).
  • unchanged.
• Example: Malonate inhibiting succinate dehydrogenase.

2. Non-competitive Inhibition:

• Inhibitor binds at an allosteric site (not the active site).
• Binds to both E and ES complex.
• Cannot be overcome by increasing substrate.
• Effect on kinetics:
  • unchanged.
  • decreases.
• Example: Heavy metals like , .

3. Uncompetitive Inhibition:

• Inhibitor binds only to the ES complex, not free enzyme.
• Effect on kinetics:
  • decreases.
  • decreases.
  • Ratio remains constant.

Comparison Table:

Enzymes are biological catalysts, mostly proteins (some are RNA molecules called ribozymes), that accelerate biochemical reactions without being consumed in the process.

General Characteristics/Properties:

1. Catalytic Efficiency:

   • Enzymes increase reaction rates by to times.

2. Specificity:

   • Highly specific for their substrates and reactions.
   • Types: absolute, group, linkage, and stereospecificity.

3. Protein Nature:

   • Most enzymes are globular proteins (except ribozymes).

4. Do Not Alter Equilibrium:

   • Enzymes speed up both forward and reverse reactions equally.
   • They only lower activation energy, not .

5. Reusability:

   • Not consumed; regenerated at the end of the reaction.

6. Active Site:

   • Have a specific region where substrate binds.

7. Sensitivity:

   • Affected by temperature, pH, and inhibitors.

8. Require Cofactors:

   • Many need metal ions (cofactors) or organic molecules (coenzymes).

Components:

• Apoenzyme: Protein part.
• Cofactor: Non-protein part (metal ion or coenzyme).
• Holoenzyme: Apoenzyme + Cofactor = active enzyme.

Activation Energy ():

Activation energy is the minimum energy required for reactant molecules to reach the transition state and convert into products. Reactions with high proceed slowly.

How Enzymes Lower Activation Energy:

• Enzymes provide an alternative reaction pathway with a lower activation energy.
• They stabilize the transition state by binding to it more tightly than to the substrate.
• This increases the number of molecules that can cross the energy barrier, increasing reaction rate.

Energy Diagram:

Energy
| Uncatalyzed (high Ea)
| /\
| / \ Catalyzed (low Ea)
| / \ /~\
| / \/ \
| S / __ P
|____
Reaction progress

Key Points:

• Enzymes do NOT change:

  • The overall free energy change ().
  • The equilibrium position.
  • The energy of substrates or products.

• Enzymes DO change:

  • The activation energy ().
  • The rate of the reaction.

Relationship (Arrhenius Equation):

Lowering increases the rate constant , thereby speeding up the reaction.

These are the two major types of secondary structure in proteins, stabilized by hydrogen bonds.

-Helix:

• Structure: A right-handed coiled/spiral structure.
• Hydrogen Bonding: Formed between the C=O of one residue and the N-H of the residue 4 positions ahead (i and i+4).
• Parameters:
  • amino acid residues per turn.
  • Pitch (rise per turn) = .
  • Rise per residue = .
• Side chains project outward from the helix.
• Hydrogen bonds are parallel to the helix axis.
• Example: Keratin, Myoglobin.
• Helix breakers: Proline and Glycine.

-Pleated Sheet:

• Structure: Extended polypeptide chains (strands) arranged side by side, giving a pleated/zigzag appearance.
• Hydrogen Bonding: Formed between adjacent strands (inter-strand), perpendicular to the chain direction.
• Types:
  • Parallel: Strands run in the same direction (N→C).
  • Antiparallel: Strands run in opposite directions (more stable).
• Distance between residues: .
• Example: Silk fibroin, -keratin.

Comparison:

Turnover Number ():

• The turnover number is the number of substrate molecules converted into product by one enzyme molecule (or active site) per unit time when the enzyme is fully saturated with substrate.

Where = total enzyme concentration.

• Units: (per second).
• Example: Carbonic anhydrase has (one of the fastest enzymes).

Catalytic Efficiency ():

• The ratio measures how efficiently an enzyme converts substrate to product at low substrate concentrations.
• It is called the specificity constant.

• Units: .

Significance:

• Higher = more efficient enzyme.
• Used to compare the efficiencies of different enzymes or the same enzyme with different substrates.
• The upper limit is the diffusion-controlled limit ( to ).
• Enzymes approaching this limit are said to have achieved catalytic perfection (e.g., catalase, fumarase).

Interpretation:

• Combines both binding affinity () and catalytic rate () into a single parameter.

Fibrous vs Globular Proteins:

Fibrous Proteins:

• Shape: Elongated, fiber-like or thread-like.
• Structure: Polypeptide chains arranged parallel along a single axis; simple, repetitive secondary structure.
• Solubility: Insoluble in water.
• Stability: Very stable, mechanically strong.
• Function: Structural and supportive.
• Examples: Collagen, Keratin, Elastin, Silk fibroin.

Globular Proteins:

• Shape: Spherical or compact/globular.
• Structure: Polypeptide chains folded into complex 3D shapes with multiple secondary structure types.
• Solubility: Soluble in water/aqueous solutions.
• Stability: Less stable; sensitive to changes in pH and temperature.
• Function: Dynamic functions (catalysis, transport, regulation).
• Examples: Enzymes, Hemoglobin, Albumin, Insulin.

Comparison Table:

Many enzymes require non-protein components for their catalytic activity. These are called cofactors.

Cofactors:

Non-protein chemical components required for enzyme activity. They are classified as:

1. Inorganic Cofactors (Metal Ions):

• Metal ions that assist in catalysis.
• Examples:
  • – Carbonic anhydrase, Carboxypeptidase.
  • – Kinases, Phosphatases.
  • – Cytochromes, Catalase.
  • – Cytochrome oxidase.

2. Organic Cofactors (Coenzymes):

• Small organic molecules, often derived from vitamins.
• Act as carriers of specific chemical groups.

Types of Coenzymes:

• Cosubstrates: Loosely bound, dissociate after reaction (e.g., NAD, NADP).
• Prosthetic groups: Tightly/covalently bound (e.g., FAD, Biotin, Heme).

Important Coenzymes and their Vitamins:

Key Terms:

• Apoenzyme: Inactive protein part.
• Holoenzyme: Apoenzyme + cofactor = active enzyme.

Anfinsen's Experiment (1961):

Christian Anfinsen conducted a classic experiment using the enzyme ribonuclease A (RNase A) to demonstrate that the primary structure of a protein determines its three-dimensional structure.

Experimental Setup:

Ribonuclease A is a small protein (124 amino acids) with four disulfide bonds ().

Steps:

1. Denaturation:

   • RNase was treated with urea (denaturant that disrupts non-covalent interactions) and -mercaptoethanol (reducing agent that breaks disulfide bonds).
   • This resulted in a completely unfolded, inactive protein.

2. Renaturation:

   • The urea and -mercaptoethanol were slowly removed by dialysis.
   • The protein spontaneously refolded into its native conformation.
   • The correct disulfide bonds reformed, and enzymatic activity was restored (~100%).

3. Control Experiment (incorrect refolding):

   • When -mercaptoethanol was removed first (before urea), disulfide bonds formed randomly, producing a scrambled, inactive protein (only ~1% activity).
   • Adding trace -mercaptoethanol allowed reshuffling to the correct native form.

Conclusions:

• The primary structure (amino acid sequence) contains all the information needed to determine the native 3D structure. This is known as Anfinsen's dogma.
• Protein folding is a spontaneous, thermodynamically driven process leading to the lowest free-energy conformation.
• Denaturation is often reversible for small proteins.

Significance:

• Laid the foundation for understanding protein folding and the thermodynamic hypothesis of folding.
• Anfinsen received the Nobel Prize in Chemistry (1972).