Enzyme Kinetics Lecture Notes

Enzyme Kinetics

• Enzyme kinetics explores the rates of enzyme-catalyzed chemical reactions and the effects of varying reaction conditions.
• Understanding enzyme kinetics reveals the catalytic mechanisms, metabolic roles, activity control, and interactions with drugs or modifiers like inhibitors or activators.
• Two main properties assessed include substrate saturation levels and the maximum reaction rate achievable by the enzyme.
• Enzymes (E) bind substrates (S) at the active site, forming an enzyme-substrate complex (ES), which transitions to an enzyme-product complex (EP) before yielding the final product (P) via a transition state (ES*).
• Simple reactions typically involve one substrate and one product but are less common than more complex two-substrate, two-product reactions, such as those catalyzed by NAD-dependent dehydrogenases like alcohol dehydrogenase.
• Enzymes may also catalyze reactions with several substrates or products; for instance, glyceraldehyde 3-phosphate dehydrogenase involves three substrates and two products.
• Enzyme kinetics can illustrate the order of substrate binding and product release, especially in enzymes like dihydrofolate reductase.
• Advanced measurement techniques involve monitoring fluorescence changes, providing insights into individual enzyme kinetics rather than averaged behavior of large populations.

Allosteric Enzymes

• "Allosteric" is derived from Greek, meaning "other space," indicating regulation where binding at one site influences another spatially distinct site.
• Allosteric enzymes are regulated by noncovalent binding to effectors or modulators, impacting their catalytic activities and controlling metabolic pathways.
• Metabolic pathways consist of interconnected reactions, where enzymatic reactions' abundance and activity influence the overall metabolic flux, or turnover rate of metabolites.
• Effector binding can be either heterotropic (where binding of a small ligand alters substrate binding at a different site) or homotropic (binding of substrate influences its own binding affinity on another subunit).
• Positive cooperativity occurs when effector binding enhances substrate binding affinity, while negative cooperativity occurs when it impedes subsequent enzyme activity.
• Allosteric enzyme kinetics follow a sigmoid growth curve rather than the hyperbolic form associated with standard Michaelis-Menten kinetics.

Notable Allosteric Enzymes

• Phosphofructokinase (PFK):

  • Key regulator in the glycolysis pathway, converting fructose-6-phosphate (F6P) to pyruvate.
  • Contains two active sites for F6P and ATP, along with a regulatory site for ATP or AMP.
  • ATP serves as a negative effector and AMP as a positive effector, modulating PFK activity based on cellular energy levels.
  • AMP stimulates glycolysis during low energy, while excess ATP inhibits it, diminishing enzyme affinity to F6P.

• Isocitrate Dehydrogenase (IDH):

  • Central to the citric acid cycle (Krebs cycle), essential for aerobic metabolism and biosynthesis of biomolecules.
  • Exists as heterodimers with catalytic and regulatory subunits, integrated into a normal operational framework.
  • Citric acid binds to the allosteric site, inducing conformational changes that facilitate substrate isocitrate binding and enzyme activation.
  • High levels of adenosine diphosphate (ADP) enhance cycle rates through additional binding alongside magnesium ions (Mg²⁺), stabilizing substrate interactions without directly altering conformation.