Enzyme Activity and Reaction Velocity

Enzyme Activity

• High activation energy (Eact) in uncatalyzed reactions results in slow reaction rates.
• Lower activation energy leads to faster reaction rates.
• Enzymes provide an alternative reaction pathway that lowers activation energy and does not affect the free energy of substrates and products.

Factors Affecting Reaction Velocity

• Substrate concentration: The rate of an enzyme-catalyzed reaction increases with substrate concentration until a maximal velocity (Vmax) is reached.
• Temperature: Increasing temperature increases the rate of both uncatalyzed and enzyme-catalyzed reactions by increasing kinetic energy and the collision frequency of reacting molecules.
  • Higher temperatures can lead to denaturation and loss of catalytic activity.
  • Human enzymes generally exhibit stability up to 45-55℃.
• pH: Most intracellular enzymes exhibit optimal activity at pH values between 5-9.
  • Extremes of pH can lead to enzyme denaturation.

Michaelis-Menten Kinetics

• Michaelis-Menten Equation:
  • Vo = initial reaction velocity
  • Vmax = maximal velocity = kcat [E]total
  • Km = Michaelis constant = (k-1 + k2)/k1
  • [S] = substrate concentration
• Assumptions:
  • Substrate concentration is greater than enzyme concentration.
  • Concentration of the ES complex does not change with time (steady-state assumption).
  • Initial reaction velocities are used in the analysis of enzyme reactions.
• Km:
  • Represents the substrate concentration at which the reaction velocity is equal to ½ Vmax.
  • Does not vary with enzyme concentration.
  • High Km indicates low affinity.
  • Low Km indicates high affinity.

Enzyme Classification by Reaction Type

• Oxidoreductases: Catalyze redox reactions.
• Transferases: Transfer moieties such as glycosyl, methyl, or phosphoryl groups.
• Hydrolases: Catalyze the hydrolytic cleavage of C—C, C—O, C—N, and other covalent bonds.
• Lyases (synthases): Catalyze cleavage of covalent bonds by atom elimination, generating double bonds.
• Isomerases: Catalyze geometric or structural changes within a molecule.
• Ligases (synthetases): Join together (ligation) of two molecules in reactions coupled to the hydrolysis of ATP.

Enzyme Structure and Function

• Active Site: Site where catalysis occurs.
  • Formed by the folding of proteins.
  • Participates in substrate binding and catalysis.
• Nonprotein Moieties: Participate in catalysis.
  • Prosthetic groups: Tightly and stably incorporated into a protein's structure by covalent bonds or noncovalent forces.
    • Eg. Pyridoxal phosphate, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), thiamine pyrophosphate, lipoic acid, biotin, transition metals – Fe, Co, Cu, Mg, Mn, Zn.
  • Cofactors: Similar to prosthetic groups, but bind weakly and transiently to their cognate enzymes or substrates.
  • Coenzymes: Small organic molecules derived from water-soluble B vitamins.
    • Eg. NAD and NADP – nicotinamide, FMN and FAD – riboflavin, Coenzyme A – pantothenic acid.
• Holoenzyme: An active enzyme with its nonprotein moiety.
• Apoenzyme: An enzyme without its nonprotein moiety.

Mechanism of Enzyme Action

• Enzyme binds substrate at the active/catalytic site.
• Active site fits the shape of the substrate.
• Association between enzyme and substrate is temporary.

Four Mechanistic Strategies During Catalysis

• Catalysis by proximity: Increases the frequency of substrate encounters.
• Acid-base catalysis: Ionizable functional groups of aminoacyl side chains act as acids or bases.
  • Specific acid or base catalysis: Participating acids or bases are protons or hydroxide ions.
  • General acid or base catalysis: Reactions responsive to all acids or bases present.
• Catalysis by strain: Creates a conformational change in an enzyme that weakens bonds.
  • Transition state intermediate: A strained conformation midway in the transformation of substrates to products.
• Covalent catalysis: Formation of a covalent bond between enzymes and one or more substrates.
  • Ping pong mechanism: First substrate is bound and its product is released prior to the bonding of the second substrate.

Enzyme Inhibitors

• Substances that decrease the velocity of an enzyme-catalyzed reaction.
  • Reversible inhibitors: Noncovalent bonds.
  • Irreversible inhibitors: Covalent bonds.
• Competitive inhibition: Inhibitor binds reversibly to the same site that the substrate would normally occupy.
  • Effect on Vmax: Unchanged.
  • Effect on Km: Increases Km.
  • Effect on Lineweaver-Burk plot: Inhibited and uninhibited reactions intersect on the y-axis at 1/Vmax and have different x-intercepts at -1/Km.
• Noncompetitive inhibition: Inhibitor and substrate bind at different sites on the enzyme.
  • Inhibitor can bind either free enzyme or the enzyme-substrate complex.
  • Effect on Vmax: Decreases apparent Vmax.
  • Effect on Km: Unchanged Km.
  • Effect on Lineweaver-Burk plot: Unchanged Km, decreased apparent Vmax.

Allosteric Enzymes

• Effectors: Bind noncovalently at a site other than the active site (allosteric site).
  • Negative effectors: Inhibitors.
  • Positive effectors: Increase enzyme activity.
• Homotropic effectors:
  • Substrates as effectors.
  • Function as positive effectors.
  • Sigmoidal curve.
  • Positive cooperativity.
  • Eg. Hemoglobin.
• Heterotropic effectors:
  • Effectors different from substrate.
  • Exemplified by feedback inhibition.
  • Eg. Phosphofructokinase-1 is allosterically inhibited by citrate (not a substrate).

Covalent Modification

• Phosphorylation and dephosphorylation:
  • Phosphorylation: Protein kinases use ATP as a phosphate donor, activating the enzyme.
  • Dephosphorylation: Phosphoprotein phosphatase deactivates the enzyme.
• Activation of zymogens:
  • Activation via proteolytic action of enteropeptidase (enterokinase) of trypsinogen → trypsin.
  • Trypsin catalyzes the subsequent conversion of numerous other pancreatic zymogens.

Enzyme Synthesis

• Cells can regulate the amount of enzyme present by altering the rate of enzyme synthesis.
• Increase or decrease of enzyme synthesis leads to alteration in the total population of active sites.
• Eg. Insulin production in response to hyperglycemia – induction of enzymes involved in glucose metabolism.

Mechanisms of Enzyme Regulation

• Regulation Event: Typical Effector, Results, Time Required for Change
  • Substrate availability: Substrate - Change in velocity - Immediate
  • Product inhibition: Reaction product - Change in Vmax and/or Km - Immediate
  • Allosteric control: Pathway end product - Change in Vmax and/or K0.5 - Immediate
  • Covalent modification: Another enzyme - Change in Vmax ad/or Km - Immediate to minutes
  • Synthesis or degradation of enzyme: Hormone or metabolite - Change in the amount of enzyme - Hours to days

Explore the fundamental concepts of enzyme activity and the factors affecting reaction rates in this quiz. Learn how activation energy, substrate concentration, temperature, and pH influence enzyme-catalyzed reactions. Test your knowledge of how enzymes operate and their importance in biological processes.