Enzyme Activity and Classification Quiz

Enzyme Activity Measurement

• Enzyme activity is measured in international units (IU): 1 IU catalyzes 1 µmol of substrate to product per minute.
• Specific activity is defined as IU per milligram of protein.

Enzyme Classification

• Enzymes are classified into seven broad categories:
  • Oxidoreductases catalyze oxidation-reduction reactions by transferring electrons (or hydrogen atoms).
  • Transferases catalyze group transfer reactions (except hydrogen atoms).
  • Hydrolases catalyze hydrolysis reactions with the addition of water.
  • Lyases/Synthases catalyze bond cleavage through elimination, resulting in double bonds or ring structures, or addition of groups to double bonds.
  • Isomerases catalyze isomerization reactions by transferring groups within molecules to yield isomeric forms.
  • Ligases catalyze bond formation through condensation reactions coupled to ATP cleavage.
  • Translocases assist in moving molecules, usually across cell membranes.

Reaction and Substrate Specificity

• Most enzymes are highly specific for the type of reaction catalyzed and the nature of the substrate.
• Examples include catalase for hydrogen peroxide and urease for urea.
• Some enzymes have broader substrate specificity, such as trypsin in protein digestion.

Active Site

• The active site is a cleft or groove where catalysis occurs.
• It contains amino acid residues that directly participate in bond making and breaking.
• The active site binds to the substrate, often with the help of cofactors or coenzymes.

Coenzymes in Enzymatic Reactions

• Cofactors are inorganic elements like calcium, iron, or copper.
• Coenzymes are organic molecules like coenzyme A.
• Coenzymes often undergo modifications during enzymatic reactions and are recycled by other enzymes.
• Coenzymes act as transient carriers of functional groups and can transport intermediates during sequences of reactions.
• Many vitamins serve as precursors of coenzymes.
• Cofactors and coenzymes can bind loosely and transiently or tightly and permanently as prosthetic groups (e.g., heme group).

Vitamin B6 and Coenzyme Derivatives

• Pyridoxal phosphate, a form of vitamin B6, is essential for amino acid synthesis, catabolism, and interconversion.
• It acts as a transient carrier of amino groups during amino acid metabolism.
• Vitamin B6 requirement increases with protein intake.
• Supplements in infant formula promote effective protein synthesis.
• B6 deficiency can lead to neurological symptoms and anemia.

Mechanism of Enzyme Action

• The enzyme's active site binds to the substrate, facilitating catalysis.
• Enzymes lower the activation energy required for the transition state.
• Enzymes increase the reaction rate without affecting the equilibrium position of the reaction.
• The binding energy released from the formation of weak interactions between the enzyme and substrate (ES complex) is denoted as ΔGB.

Enzyme Kinetics: Michaelis-Menten Kinetics

• The initial rate (V0) of an enzymatic reaction is described by the Michaelis-Menten equation:
  • V0 = Vmax * [S] / (Km + [S])
• Vmax represents the maximum reaction rate when the enzyme is saturated with substrate ([ES] = [Etotal]).
• Km is the substrate concentration at which the initial rate is half of its maximal rate.
• Km reflects the dissociation constant for the ES complex (E + S) and indicates the enzyme's affinity for its substrate.
  • A lower Km indicates a higher affinity for the substrate and vice versa.
• The equation is derived from the initial rate of a simple enzymatic reaction, assuming that the rate of the first step (E+S → ES) is much slower than the rate of the second step (ES → E + P).

Lineweaver-Burk Double Reciprocal Plot

• Taking the reciprocal of both sides of the Michaelis-Menten equation yields the Lineweaver-Burk equation:
  • 1/V0 = Km/(Vmax*[S]) + 1/Vmax
• This equation represents a linear equation (y = mx + c).
• The slope of the Lineweaver-Burk plot is Km/Vmax, and the y-intercept is 1/Vmax.

Turnover Number (Kcat)

• Kcat represents the number of substrate molecules converted to product per unit time when the enzyme is saturated with substrate.
• Kcat is determined by the rate constant of the step ES → E + P, represented by k2.
• If ES → E + P is the rate-limiting step, then k2 = Kcat.
• The unit of Kcat is s-1.

Catalytic Efficiency (Kcat/Km)

• Kcat/Km reflects the catalytic efficiency of an enzyme, combining both the rate of catalysis (Kcat) and the affinity for the substrate (Km).

Nature and Inhibition of Enzymes

• Competitive Inhibition (reversible):
  • The inhibitor competes with the substrate for the active site.
  • The effect can be overcome by increasing substrate concentration.
  • The inhibitor does not inactivate the enzyme.
  • Vmax remains unchanged.
  • Km increases (decreased substrate affinity).
  • The Lineweaver-Burk plot shows an increased slope.
• Noncompetitive Inhibition (reversible):
  • The inhibitor binds to a site distinct from the active site, affecting both E and ES complex.
  • The inhibitor can inactivate the enzyme.
  • Vmax decreases.
  • Km remains unchanged.
  • The Lineweaver-Burk plot shows an increased slope and a higher y-intercept.
• Irreversible Inhibition:
  • The inhibitor binds strongly to the active site, often through covalent bond formation.
  • Suicide inactivators are irreversible inhibitors that initially perform the first few steps of the reaction but cannot be converted to product, leading to permanent enzyme inactivation.

Regulation of Enzymatic Reactions

• Allosteric Regulation:
  • Allosteric enzymes are typically larger and have at least two subunits (catalytic and regulatory).
  • They interact with inhibitors and activators, which are usually small metabolites or cofactors.
  • The binding of the modulator (inhibitor or activator) causes conformational changes in the enzyme.
  • These changes can affect catalytic activity.
  • Aspartate transcarbamoylase is an example of an allosteric enzyme, regulated by CTP (inhibitor) and ATP (activator).
• Reversible Covalent Modification:
  • This involves the addition or removal of phosphoryl groups, affecting the structure and catalytic activity of enzymes.
  • Kinases add phosphate groups, while phosphatases remove them.
• Proteolytic Cleavage of Enzyme Precursor:
  • Digestive enzymes are often synthesized and stored as inactive precursors called zymogens or proenzymes.
  • Proteolytic cleavage activates these precursor enzymes.

Acute Pancreatitis

• This condition arises when pancreatic enzymes become activated within the pancreatic ductal system.
• The activated enzymes damage proteins on the cell surface and blood vessels.
• Serum contains natural inhibitors for trypsin (antitrypsin).
• Normally, pancreatic enzymes remain inactive until reaching the duodenum.

Enzymes in Health and Disease

• Numerous enzymes are involved in various important physiological processes (e.g., digestion, metabolism, and energy production).
• Enzyme deficiencies or malfunctions can lead to diseases.
  • For example, deficiencies in lysosomal enzymes can cause genetic disorders.
  • Enzyme inhibitors are widely used as therapeutic agents.