Enzyme Kinetics and Inhibition Notes

Enzymes: Kinetics and Inhibition

Enzyme Kinetics

• Reaction rates are measured by observing the change in concentration of either product appearance or substrate disappearance per unit of time, typically per second.
• As the reaction proceeds, more substrate is converted to product over time.
• Product inhibition or enzyme instability can result in a non-linear plot of concentration vs. time.
• The rate of an enzymatic reaction depends on several factors:
  • pH: Most mammalian enzymes are most active at a pH of approximately 7.0, while lysosomal enzymes and pepsin require an acidic pH.
  • Temperature: Reaction rates typically increase about 2-fold for every 10°C rise in temperature because more molecules reach a higher energy state. However, many enzymes denature at higher temperatures.
  • Enzyme Concentration
  • Substrate Concentration

Vmax and KM

• $V_{max}$ is the maximum reaction velocity.

• $K<em>M$ is the substrate concentration [S] at which the reaction velocity is half of $V</em>{max}$.

• Many enzymatic reactions follow Michaelis-Menten kinetics, described by the equation:
  $V<em>0 = [0.5ex] \frac{V</em>{max} [S]}{[S] + K<em>M}$, where $V</em>0$ is the initial reaction rate.

• When [S] = $K<em>M$, $V</em>0 = V_{max} / 2$.

• $K<em>M$ relates to the steepness of the curve, and $V</em>{max}$ relates to the height of the curve in a reaction rate plot.

Linear Plots of Kinetic Data

• Kinetic data is often plotted in double reciprocal form.
• Plot 1/V vs. 1/[S].
• $1/V<em>{max}$ and $-1/K</em>M$ are obtained from the intercepts with the Y and X axes, respectively.

Hexokinase and Glucokinase

• Hexokinase and glucokinase catalyze the same reaction but have different $K_M$ values.
• Both enzymes use the energy of ATP to form glucose-6-phosphate:
  $Glucose + ATP \longrightarrow glucose-6-phosphate$
• Hexokinase has a smaller $K_M$ than glucokinase and is more efficient at lower glucose concentrations.
• Glucokinase exhibits more complex kinetics, displaying an “S-shaped” or sigmoidal curve.

Isozymes

• Isozymes or isoenzymes differ in amino acid structure but catalyze the same reaction.
• Isozymes can be distinguished by properties such as electrophoretic mobility.
• Isozymes permit fine-tuning of metabolism:
  • Most cells contain hexokinase and can utilize glucose when concentrations are low.
  • Liver cells contain glucokinase, which allows the liver to utilize glucose when blood glucose levels rise, storing it as glycogen and converting excess glucose to fat.

Lactate Dehydrogenase (LDH) Isozymes

• LDH is a tetrameric enzyme with two isozymic polypeptide chains, resulting in 5 different tetramers.
• The heart primarily contains the H form (H4 and some H3M).
• Skeletal muscle and liver mostly contain M4.
• LDH-1 or H4 functions best under highly aerobic conditions and is the major form in the adult heart.
• LDH-5 or M4 functions best with lower [O2].
• LDH-5 is the major form in the mammalian heart during fetal development.

Enzyme Inhibition

• Enzyme inhibition is a physiological control mechanism that regulates metabolic activities to meet the needs of the cell.
• Many therapeutic substances are enzyme inhibitors:
  • Aspirin inhibits cyclooxygenase, blocking prostaglandin synthesis.
  • Penicillin inhibits bacterial growth by blocking a step in cell wall synthesis.
• Toxic chemicals can act as enzyme inhibitors:
  • Organofluorophosphates are toxic nerve gases that inhibit acetylcholinesterase.

Types of Enzyme Inhibition

• Reversible (occurs with removal of the inhibitor)
  • Competitive
  • Noncompetitive
• Irreversible
  • Usually modify amino acid side chains via irreversible chemical reactions.
  • Sometimes function by very tight binding to the enzyme without reacting chemically.
  • Includes processes that completely denature the enzyme protein.

Two Types of Reversible Inhibition

• A competitive inhibitor binds at the active site, preventing substrate binding.
• A noncompetitive inhibitor does not prevent substrate binding but interferes with catalysis, decreasing the catalytic efficiency of the enzyme.

Competitive Inhibition

• With competitive inhibition, the enzyme can bind either the substrate or the inhibitor. If the enzyme binds the inhibitor, it cannot react with the substrate.
• The inhibitor decreases the reaction rate, especially at low substrate concentrations.

Kinetics of Competitive Inhibition

• The kinetics of the reaction are affected by the relative concentrations of substrate and inhibitor.
• As [I] increases, a higher [S] is required to attain the same reaction rate.
• Sufficiently high [S] can completely relieve competitive inhibition.

Kinetics of Noncompetitive Inhibition

• The inhibitor can bind either to the free enzyme (E) or to the enzyme-substrate complex (ES). The EI complex can still bind substrate.
• If the ES complex has I bound to it, the reaction cannot occur.
• Even at very high [S], $V_{max}$ cannot be attained.

Competitive vs. Noncompetitive Inhibition

• A competitive inhibitor increases $K<em>M$ but has no effect on $V</em>{max}$.
• A noncompetitive inhibitor decreases $V<em>{max}$ but does not affect $K</em>M$.

Inhibition of Proteases

• Cells can be protected from proteolytic enzymes by specific protease inhibitors.
• Trypsin is inhibited by a trypsin inhibitor, also produced in the pancreas.
• The inhibitor is a very tight-binding substrate analog and thus a competitive inhibitor.
• α-1 antitrypsin is a plasma protein that protects the lungs from damage by elastase.

Irreversible Inhibition

• Aspirin is an irreversible inhibitor of cyclooxygenase (aka prostaglandin synthase). It acts by covalently transferring an acetyl group to a serine residue (Ser 530) on the enzyme.
• Binding of other cyclooxygenase inhibitors, such as ibuprofen, to the active site does not involve covalent modification. Ibuprofen is thus a reversible inhibitor of the enzyme.