Enzymes and biocatalysis

Enzymes are proteins that act on substrates, catalyzing chemical reactions within the cell. Enzymes are specific in the sense that each enzyme only reacts with a few closely related substrates. Some enzymes require cofactors (biotin, lipoamide, cobalamin) to function properly. Enzymes can become denatured by changes in temperature or pH. Enzymes are classified as oxidoreductases, transferases, hydrolases, lyases, ligases, and isomerases, based on the type of reaction they catalyze. Enzyme kinetics is the study of enzyme reaction rates, which are determined using the Michaelis-Menten and Lineweaver-Burk equations. These equations can also be used to evaluate how different types of enzyme inhibitors affect the reaction rate. Enzyme deficiencies can result in severe diseases such as Lesch-Nyhan syndrome, Gaucher disease, and phenylketonuria.

• Complex proteins that catalyze chemical reactions
• Act on substrates that can either be cleaved or joined to form a new product (e.g., carbonic anhydrase enzyme → CO₂ + H₂0 ⇄ H₂CO₃)
• Essential for life; if enzymes did not exist, cellular reactions would not occur fast enough to sustain life.
• Enzyme deficiencies can result in severe diseases (e.g., Lesch-Nyhan syndrome).
• Enzyme name is usually based on the reaction catalyzed plus the suffix “-ase”: e.g., for the enzyme that adds hydroxyl groups (OH^-) is formed as follows: hydroxyl + -ase → hydroxylase

General characteristics

• Active site: binding site for a specific substrate on a specific enzyme
• Specificity
  • Enzymes are highly specific for their substrate and product.
  • Some exceptions include proteases that break down proteins to peptides in the digestive system.
• Rate: enzymes catalyze reactions by a factor of 10⁶–10¹¹
• Coenzymes
  • Many enzymes require coenzymes (e.g., biotin) that allow them to perform their action on a substrate.
  • Usually small organic molecules derived from metal ions or vitamins
• Thermodynamics
  • Enzymes do not affect the energy level of substrates or products (free energy released remains the same).
  • Enzymes are able to decrease the energy of activation required to start a reaction.
  • The velocity of enzymatic reactions increases with temperature (up to 37^o C in humans).
• pH
  • Each enzyme has a specific pH at which it can achieve maximum velocity (V_max).
  • Alterations in pH can cause denaturation of enzymes (specific to each enzyme).
  • Example: Pepsin works best in acidic environment like the stomach (pH ∼1.5–2) and it is inactivated in the duodenum when bicarbonate is released from the pancreas, increasing the pH to > 7.

Gibbs energy

Energy (∆G) for enzymatic reactions usually comes from the break down of ATP or GTP bonds (hydrolysis). Enzymatic reactions can occur spontaneously or nonspontaneously. The following are relationships between energy and enzymatic activity.

• Exergonic: Energy (∆G) < 0: Reactions can occur spontaneously (often irreversible).
• Endergonic: Energy (∆G) > 0: Reactions require energy to occur (from ATP or GDP).
• Balanced reaction: Energy (∆G) = 0: The reaction is at equilibrium (reversible).

For more information, see “Vitamins.”

Michaelis-Menten kinetics

Description of an enzymatic reaction

• Enzymatic reactions with a hyperbolic curve (most common, e.g., alcohol dehydrogenase in ethanol oxidation): E + S ⇄ ES → E + P
  • [E] = enzyme
  • [S] = substrate
  • [P] = product
  • [V] = velocity
• A sigmoid curve indicates cooperativity (e.g., oxygen binding to hemoglobin)

Enzymatic reactions with a sigmoidal kinetic are indicative of cooperative binding (e.g., oxygen to hemoglobin).

Michaelis-Menten equation

• Equation: v = V_max [S] / (K_m + [S])
• Maximum velocity (Vmax)
  • Maximum rate at which an enzyme can catalyze a reaction
  • Directly proportional to the enzyme concentration
    • The only way to ↑ V_max is to increase [E] (cells achieve this, e.g., by increasing gene expression of a given enzyme)
    • ↑ Enzyme concentration → ↑ V_max
    • Noncompetitive inhibitors → ↓ [E] → ↓ V_max
• Michaelis constant: (K_m): the substrate concentration at which half of the active sites of the enzymes are bound to the substrate
  • Reaction velocity is ½ of V_max when the Michaelis constant concentration is reached
  • Inversely proportional to the affinity of the enzyme for the substrate: ↑ enzyme affinity → ↓ K_m
• Michaelis-Menten plot: a way of modeling Michaelis-Menten enzyme kinetics that relates the concentration of the substrate to reaction rate

Lineweaver-Burk equation and plot

Equation

• The Lineweaver-Burk equation is a double reciprocal of the Michaelis-Menten equation, where V = V_max [S] / K_m + [S] (if [E] remains constant), becomes 1 / v = K_m / V_max× 1/[S] + 1 / V_max.
• Represents enzyme kinetics in a linear graph rather than a hyperbola
• Equation is particularly important to determine the effect of drugs on enzymes

Plot

• Intercept with y-axis: : 1/V_max, the further from zero, the lower V_max
• Intercept with x-axis: : 1/-K_m : the closer to zero, the lower the affinity and the higher the K_m
• Slope: K_m/V_max

Very efficient and kompetent: On the Lineweaver-Burk plot, V_max usually represents the efficacy of a drug on the y-axis and K_m represents the potency on the x-axis.

Drug-response dynamics

For details, see “Pharmacodynamics.”

Uncompetitive inhibitors are enzyme inhibitors that bind to the enzyme-substrate complex, decreasing K_m and V_max.

Kompetitive Inhibition: K_m Increases and V_max remains unchanged. Nonkompetitive Inhibition: No K_m change and V_max decreases.

Only Companions meet on the waY: Competitive inhibitors meet on the Y-axis (same V_max), noncompetitive do not.

• Glucose-6-phosphate dehydrogenase deficiency
• Chronic granulomatous disease
• Lesch-Nyhan syndrome
• Von Gierke's disease
• Pompe's disease
• Cori's disease
• Andersen's disease
• McArdle's disease
• Galactosemia
• Hereditary fructose intolerance
• Essential fructosuria
• Medium chain Acyl-CoA dehydrogenase deficiency
• Mucopolysaccharidoses
• Gaucher disease
• Krabbe disease
• Tay-Sachs disease
• Fabry disease
• Metachromatic leukodystrophy
• Niemann-Pick disease
• α1-antitrypsin deficiency
• Mitochondrial myopathies
• Alkaptonuria
• Homocystinuria
• Phenylketonuria
• Histidinemia