BISC_312_Lecture_11_2021

Enzyme Kinetics

Overview

  • Focus on the kinetics of enzymes which involves studying how enzymes facilitate biochemical reactions.

Michaelis-Menten Kinetics

Derivation Basics

  • While the specifics of deriving the Michaelis-Menten (M-M) equation may not be tested, understanding how to interpret and apply values like Km and Vmax is essential.

  • Referenced videos provide insights into these values and their implications.

Measuring Reaction Rates

  • Method of studying enzymatic reaction rates:

    • Monitor the disappearance of substrate (S) over time with varying concentrations.

    • Use the formula: V = -ΔS/Δt = kF [S].

    • Observations:

      • Reaction velocity (V) slows down as S is converted to product (P).

      • Reverse reaction becomes significant as P accumulates, leading to equilibrium (VF = VR).

      • V0 is determined at very early time points when [S] has not significantly decreased.

M-M Curve Interpretation

  • Different regions of the Michaelis-Menten curve:

    1. At very low [S] (much less than Km), V0 is approximately equal to kF[S], indicating first-order kinetics.

    2. At [S] = Km, V0 is half of Vmax (V0 = Vmax/2).

    3. At very high [S] (much greater than Km), V0 approaches Vmax, indicating zero-order kinetics, and is independent of [S].

    • Equation: V0 = Vmax[S] / (Km + [S]).

Determining Km and Vmax

  • Utilize a reciprocal plot of the M-M curve:

    • Resembles the linear equation y = mx + b.

    • Key points:

      • x-intercept: -1/Km

      • y-intercept: 1/Vmax

      • slope: Km/Vmax.

  • Reaffirm that Km is a concentration value, always positive.

Enzymatic Inhibition

Types of Inhibition

  • Reversible Inhibition: Non-covalent, effective binding; can be alleviated.

  • Irreversible Inhibition: Form covalent bonds with enzymes, permanently inactivating them.

Modes of Reversible Inhibition

  1. Competitive Inhibition:

    • Inhibitor (I) competes with substrate (S) for the active site.

    • Increases Km but does not change Vmax.

    • Higher [S] can overcome inhibition.

    • Ki is the dissociation constant of the inhibitor (Ki = [E][I]/[EI]).

  2. Uncompetitive Inhibition:

    • Inhibitor binds only to the enzyme-substrate complex (ES).

    • Lowers both Km and Vmax; high [S] cannot overcome inhibition.

  3. Mixed Inhibition:

    • Both E and I can bind simultaneously, forming an ESI complex that cannot produce P.

    • Vmax is reduced; Km may increase or remain unchanged.

    • High [S] does not overcome this inhibition.

Examples of Inhibitors

  • Captopril: An inhibitor for angiotensin-converting enzyme (ACE), used in hypertension treatment.

    • Competitive inhibitor, binds tightly to the active site (25,000x more than normal substrate) with Km of 52 µM vs Ki of 1.7 nM.

Regulation of Enzyme Activity

Need for Regulation

  • Enzymes are assembled in pathways; not all need to operate at peak activity simultaneously.

  • Mechanisms of regulation include:

    1. Substrate-level Control: Interaction of substrates/products affects enzyme activity.

    2. Feedback Control: End products inhibit earlier steps in the pathway.

    3. Allosteric Regulation: Conformational changes in enzymes alter activity.

Substrate-level Control

  • Interaction between substrates and products can lead to inhibition or enhancement of product formation.

  • Example: High levels of glucose-6-phosphate inhibit hexokinase.

Allosteric Enzymes

Characteristics

  • Multi-subunit proteins; exhibit different binding and catalytic activities.

  • Behavior reflects a transition between two states:

    • T (tense) state: lower catalytic activity, compact form.

    • R (relaxed) state: higher catalytic activity, expanded form.

Cooperative Effects

  • Low [S] leads to low affinity for further S binding, while high [S] increases the affinity and catalytic activity markedly.

  • This results in a steep increase in catalytic rate with small changes in [S].

Example: ATCase

  • Aspartate Transcarbamoylase (ATCase) regulates pyrimidine biosynthesis, with significant control enabled by CTP (inhibitor) and ATP (activator).

  • CTP binding favors the T state, inhibiting activity, while ATP promotes the R state, stimulating activity.

Feedback Inhibition

  • CTP modulates the equilibrium between T and R states, effectively reducing the affinity of ATCase for its substrates when in abundance.