Enzyme Kinetics Lecture Notes

Study Notes

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" 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.

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.