Week 5 enzyme kinetic 1

Vmax: Maximum attainable rate of reaction with a fixed concentration of enzyme.
Km (Michaelis constant): Amount of substrate required for half-maximal reaction rate; indicates enzyme-substrate binding affinity.
Kcat: Turnover number; maximum amount of substrate converted per unit time.
Kcat/Km: Ratio known as 'catalytic efficiency' or 'specificity constant'.

The model describes the interaction of enzyme and substrate, forming an enzyme-substrate complex (E-S complex) in a reversible manner.
Governed by two rate constants that yield the formation rate of the E-S complex, favoring the formation due to excess substrate.
The equilibrium is quickly established between free enzyme and free substrate plus the E-S complex.
Catalysis occurs as the product binds to the active site, eventually leading to product release, described by the rate constant K2.
As product concentration increases, enzyme-product complexes may also form, affecting reaction rates in reversible processes.

With fixed substrate concentration, reactions will produce product until equilibrium is reached, where the rates of forward and reverse reactions equalize.
Initial reaction rate values represent the product formed per unit time in the early phase of the reaction, where the reverse reaction is negligible.
Steady State Assumption: Concentration of free enzyme (E) and E-S complexes remain constant; substrate concentration typically remains in excess.
This assumption allows simplification of the Michaelis-Menten model.

The rate depends on substrate binding and product release, and initially is first-order with respect to substrate concentration.
At low substrate concentrations, the reverse reaction is negligible.
The model allows derivation of kinetic constants providing insights into enzyme efficiency and specificity.

Typical Michaelis-Menten Plot: Conducted by incubating fixed enzyme quantity with varying substrate concentrations.
As substrate concentration increases, the rate of reaction (RoR) will initially rise almost proportionate to substrate concentration, showing first-order behavior.
At higher substrate concentrations, a plateau (approach to Vmax) is reached as enzymes become saturated.

At near Vmax, nearly all enzyme exists in the E-S complex, and the rate of reaction becomes zero-order as it no longer depends on substrate concentration.
Vmax serves as a measure of catalytic power, but it is essential to compare it with Kcat for understanding efficiency, as Vmax alone isn't a conclusive measure.
Kcat provides the turnover rate per enzyme molecule, facilitating comparisons across different enzyme systems.

Low Km Value: Indicates a high binding affinity between enzyme and substrate; requires less substrate for half-saturation.
High Km Value: Indicates a lower binding affinity; more substrate is required for half-saturation.
Km is critical for understanding effective catalysis; however, its correlation with binding affinity is limited by factors like dissociation rates and catalytic efficiency.

Catalytic efficiency is defined by the ratio Kcat/Km, indicating the efficiency with which substrate is converted to product.
Effective enzyme catalysis is influenced by diffusion rates and can exhibit high efficiencies (10^8 - 10^9 s^-1 M^-1 considered kinetic perfection).
The Michaelis-Menten equation allows quantification of the rate of reaction based on substrate concentration.

The Lineweaver-Burk (LB) plot transforms the hyperbolic MM curve into a linear form, allowing calculation of Vmax and Km values.
The LB plot is particularly effective at analyzing the effects of enzyme inhibitors.
Limitations exist due to data bias from the reciprocal scaling leading to inaccuracies in low substrate concentration measurements.

Competitive Inhibition:
- Competes directly with substrate for binding to the active site.
- Binding is reversible; increasing substrate concentration can outcompete the inhibitor.
- Km increases (lower affinity), but Vmax remains unchanged.
Non-Competitive Inhibition:
- Inhibitor binds to a different site than the active site, stabilizing an inactive enzyme conformation.
- Substrate can still bind, but the inhibitor prevents catalysis from occurring; Vmax decreases while Km remains unchanged.

Involves tight binding to the enzyme, causing permanent inactivation with slow dissociation.
Commonly occurs with nerve agents; noted for interactions that form stable covalent bonds with active site residues.

Denaturating Factors: High temperature, extreme pH, denaturing reagents, and high salt concentrations can lead to protein unfolding, aggregation, and loss of function.
Renaturation is difficult post-denaturation, especially for large proteins.

Correct folding is driven by weak interactions and mediated by chaperones, which assist in achieving the native conformation post-translationally.
Chaperones and isomerases are integral to correct protein folding, essential in biological systems.

Changes in pH and temperature affect enzyme activity and overall catalytic performance, adhering to an optimal range for function.
Optimal pH varies (cytoplasmic vs. lysosomal vs. digestive enzymes) and can influence enzyme-substrate interactions.