Enzymes: Biological Catalysts

• Enzymes are biologic catalysts increasing the rate of noncatalyzed reactions.

Biologic Catalysts

• Enzymes are proteins, except for ribozymes (catalytic RNAs).
• Enzymes catalyze desired reactions with high specificity, reducing side reactions.
• Enzymes remain unchanged but may be temporarily altered during catalysis.

Enzyme Energetics

• Chemical reactions or physical processes are accompanied by a change in free energy (ΔG).
• Reactions proceed spontaneously with a negative ΔG (decrease in G).
• ΔG depends on enthalpy (H) and entropy (S): ΔG = ΔH – TΔS.
• Heat release and increased disorder contribute to reaction spontaneity.
• ΔG indicates whether a reaction proceeds forward from equilibrium, not the rate.
• Activation energy determines reaction rate, even for spontaneous reactions.
• Enzymes decrease activation energy, increasing reaction rate in both directions.
• Enzymes cannot force nonspontaneous reactions.

Common Intermediates and Coupling

• Nonspontaneous reactions occur when coupled with reactions with a negative ΔG through a common intermediate.
• The product of one reaction is the reactant in a second reaction, coupling them.

Spontaneity and Product Removal

• Standard free energy changes are measured under standard conditions (1 mol/L reactants, 25°C, 1 atm, pH 7).
• Actual free energy change depends on standard tendency and mass action effect.
• Fuel addition and waste removal in metabolic pathways ensure spontaneity.

Metabolic Pathways

• Metabolic pathways are coupled reactions with common intermediates.
• Free energy changes are summative, ensuring a negative overall ΔG in catabolic pathways.
• Pathways achieve catabolism (energy extraction/storage), anabolism (synthesis), and digestion (molecule degradation without energy storage).

Enzyme Nomenclature

• Enzymes are classified by the reaction type they catalyze:
• Oxidoreductases: Transfer electrons.
• Transferases: Transfer functional groups.
• Hydrolases: Cleave bonds via water addition.
• Lyases: Add or remove water, ammonia, or CO2.
• Isomerases: Interconvert isomeric forms.
• Ligases: Form new covalent bonds using ATP.

Coenzymes

• Enzymes require nonprotein cofactors (coenzymes); tightly bound ones are prosthetic groups.
• The apoenzyme lacks the prosthetic group, while the holoenzyme is fully functional.
• Coenzymes, often vitamins, can be regenerated through coupling.
• Metal ions also serve as cofactors.

Active Site Properties

• Enzymes lower activation energy by binding substrates in a specific configuration within a protected active site.
• Active sites are often clefts within the enzyme tertiary structure.
• Active site amino acid residues are often distant in the primary structure.
• Vitamin deficiencies correlate with enzyme cofactor function.
• Vitamin C deficiency (scurvy) causes weak connective tissue due to deficient proline hydroxylation in collagen.

Induced Fit

• Enzyme active sites are not rigid; they undergo conformational changes upon substrate binding (induced fit).
• Induced fit is necessary to exclude water and prevent side reactions like hydrolysis.

Amino Acid Composition

• Active site amino acids form ionic, hydrogen bonds, and hydrophobic interactions with substrate.
• Enzyme activity depends on pH, temperature, and ionic strength due to these bonds.
• The amino acids forming the active site are highly conserved across species.

Transition State

• Substrates adopt an activated "transition" state upon binding to the active site.
• Transition state analogs are effective enzyme inhibitors due to high affinity, blocking the reaction.

Analytic Methods

• These methods are used to study active sites, enabling specific drug development:
• Affinity labels: Substrate analogs that react with active site amino acids.
• X-ray diffraction: Reveals spatial relationships with bound substrate or transition state analog.
• Site-directed mutagenesis: Amino acid substitutions identify crucial active site residues.

Multisubstrate Reactions

• When two or more substrates are involved in a reaction, the binding order can be random or sequential.
• Sequential: both substrates must bind for the reaction to take place to occur, can occur in a specific or random order.
• Ping-pong: Each substrate binds and reacts in turn, releasing a product before the next substrate binds, creating an intermediate form.

Kinetics

• Reaction velocity is measured under initial conditions with high substrate concentration to avoid reverse reaction interference.
• Enzyme amounts are expressed in units (1 unit = µmol/min) or international units per liter (IU/L).

Reaction Order

• Reaction order is determined by the number of substrates affecting reaction rate:
• Zero order: Enzyme is saturated; rate is independent of substrate concentration.
• First order: Rate is directly proportional to substrate concentration.
• Second order: Rate is proportional to the concentration of two substrates.

Michaelis-Menten Kinetics

• The Michaelis-Menten model expresses the kinetic properties of enzymes.
• Single substrate (S) binds reversibly to form ES complex (E + S ⇌ ES → E + P).
• ES can form product (P) or break down to enzyme and substrate without reacting.
• Enzyme activity plotted against substrate concentration forms a rectangular hyperbola.
• Activity is linearly proportional (first order) at low substrate concentrations.
• At saturating substrate concentrations (zero order), activity reaches maximal velocity (Vmax).
• Michaelis constant (Km) is the substrate concentration at half Vmax.
• Km is an inverse measure of enzyme-substrate affinity.
• Vmax is directly proportional to enzyme concentration.
• Lineweaver-Burk plot (double reciprocal plot of 1/v vs. 1/S) yields a linear representation:
• Intersection with 1/S axis equals -1/Km.
• Intersection with 1/v axis equals 1/Vmax.

Inhibition

• Inhibition by nonphysiologic agents (drugs, toxins) results in complete enzyme inactivation.
• Inhibition by metabolites (allosteric inhibitors) causes a gradual activity reduction.

Competitive Inhibitors

• Competes with the substrate for the active site.
• Forms enzyme-inhibitor complex, preventing the substrate from binding.
• Competitive inhibitors are often substrate analogs.
• Methotrexate, an antineoplastic drug, competes with folic acid, inhibiting dihydrofolate reductase and blocking DNA synthesis.
• Competitive inhibition increases Km but does not change Vmax.

Noncompetitive Inhibitors

• Binds reversibly to the enzyme at a site away from the active site.
• This still allows for the substrate to bind normally.
• The enzyme is fully inactivated when the inhibitor is bound.
• Noncompetitive inhibition reduces Vmax but doesn't affect Km.

Optimal Conditions

• Enzymes have an optimum pH.
• Enzymes denature at extreme pH values.
• Ionization changes with different pH values, which influence the affinity of the enzyme for its substrate.
• Enzyme activity varies among sources in temperature optima, cellular location, Vmax, Km, and amino acid composition
• Active sites are highly conserved.

Irreversible Inhibitors

• Permanently inactivate enzymes.
• The only way for reversing the inactivation of the enzyme is the synthesis of new enzyme protein by the cell.
• Enzymes demonstrate temperature optimum, at which an increased rate is eventually slowed and then reversed as denaturation destroys structure.
• Aspirin inhibits cyclooxygenase I irreversibly.
• Ibuprofen inhibits cyclooxygenase I reversibly.

Regulation

• Regulation is the response of enzyme activity to physiological changes, structured in specialized sites.
• External signals are transmitted as second messengers from hormone-receptor binding.
• Internal signals are metabolic intermediates.
• External signals transmit via covalent modification; internal signals transmit via allosteric regulation.

Covalent Modification

• Protein kinase phosphorylates serine, threonine, or tyrosine in the regulated enzyme.
• Protein phosphatase dephosphorylates the regulated enzyme to restore the nonphosphorylated form.
• Phosphorylation can activate or inactivate the enzyme.
• Intracellular signaling uses a cascade mechanism to amplify hormonal signals sequentially.

Allosteric Regulation

• Allosterism results from effector molecule binding, increasing or decreasing enzyme activity.
• To be regulated by an allosteric effector, the enzyme must first demonstrate cooperativity.
• Positive cooperativity: Ligand binding on one subunit increases ligand binding on adjacent subunits, producing an S-shaped curve.
• Sequential mechanism: Change in activity occurs one subunit at a time.
• Concerted mechanism: Change in activity occurs simultaneously in all subunits.

Cellular Regulatory Strategies

• Multiple regulatory strategies reflect wide adaptations within cells and the body.

Metabolic Pathway Regulation

• Cells regulate pathways through compartmentation, gene regulation, covalent modification, and allosteric regulation.
• Compartmentation: Permanent regulation via physical separation
• Gene regulation: Long-term regulation, slow response.
• Covalent modification: Rapid regulation.
• Allosteric regulation: Instantaneous regulation.

Proenzymes (and Prohormones)

• Inactive storage forms activated by proteolytic removal.
• Examples: complement/clotting pathways, proinsulin.

Isoenzymes

• Alternative forms of same enzyme activity in varying tissue proportions.
• They differ in amino acid composition/sequence and multimeric structure; they have similar structures.
• Isoenzyme expression in tissues determines regulation
• Isoenzymes identified via electrophoresis.

Diagnostic Enzymology

• Tissue damage releases enzymes into the serum.
• This occurs in proportion to the amount of damage that occurred.
• The particular enzymes released can be isoenzyme forms that are tissue specific.