Enzymes II: Kinetics, Inhibition, and Metabolic Regulation

Course and Lecture Fundamentals

• Course Code: 4BBY1013 Biochemistry

• Module Title: Enzymes II

• Institution: Faculty of Life Sciences & Medicine, Department of Biochemistry

• Lecturer: Dr. Gian De Nicola

Enzyme Inhibition and Kinetic Parameters

• Michaelis-Menten Equation: The velocity of an enzyme-catalyzed reaction is expressed by the formula:     $V = \frac{V_{max}[S]}{[S] + K_m}$

  • $V$: Velocity of the reaction.

  • $V_{max}$: The maximum velocity attained by the system at saturating substrate concentrations.

  • $K_m$: The Michaelis constant, representing the substrate concentration at which the reaction velocity is half of $V_{max}$.

  • $[S]$: Concentration of the substrate.

• Lineweaver-Burk (Double-Reciprocal) Plot: This linear representation of the Michaelis-Menten equation is used to determine kinetic constants graphically:     $\frac{1}{V} = \frac{K_m}{V_{max}} \times \frac{1}{[S]} + \frac{1}{V_{max}}$

  • The y-intercept is equal to $\frac{1}{V_{max}}$.

  • The x-intercept is equal to $-\frac{1}{K_m}$.

  • The slope of the line is equal to $\frac{K_m}{V_{max}}$.

Competitive Inhibition

• Mechanism: Competitive inhibitors compete directly with the substrate for binding to the enzyme's active site.

• perturbing the recognition between the substrate and the enzyme

• doesn’t change the rate at which the substrate s converted into the correct product when the enzyme recognises the correct substrate

• Effect on Kinetics:

  • Apparent $K_m$ ($K_{m,app}$): Competitive inhibitors increase the apparent value of $K_m$

  • $V_{max}$: Competitive inhibitors do not alter the $V_{max}$. If the substrate concentration is increased sufficiently, the inhibitor can be displaced, and the enzyme can still reach its maximal catalytic rate.

• Lineweaver-Burk Characteristics:

  • The slope increases ($\frac{K_{m,app}}{V_{max}}$) as inhibitor concentration ($[I]$) increases.

  • The lines for different inhibitor concentrations intersect at the same y-intercept ($\frac{1}{V_{max}}$) but have a larger Km.

Non-Competitive Inhibition

• Mechanism: Non-competitive inhibitors do not bind to the active site. Instead, they interfere with the catalytic mechanism in another way, often by binding to an allosteric site or a different region of the enzyme, regardless of whether the substrate is bound.

• Effect on Kinetics:

  • $V_{max}$: These inhibitors decrease the $V_{max}$. Even at infinite substrate concentration, the enzyme cannot reach its full catalytic potential because the catalytic machinery itself is impaired.

  • $K_m$: Non-competitive inhibitors do not alter the $K_m$. The substrate's affinity for the active site remains unchanged.

• Lineweaver-Burk Characteristics:

  • The y-intercept ($\frac{1}{V_{max}}$) increases as inhibitor concentration increases.

  • The x-intercept ($-\frac{1}{K_m}$) remains constant, meaning all lines intersect at the same point on the x-axis.

• Example: Cyanide:

  • Cyanide inhibits Cytochrome c oxidase.

  • It functions by interfering with the transport of electrons within the enzyme machinery.

Irreversible Inhibition

• Definition: Irreversible inhibitors bind so strongly (often through covalent bonds) that the enzyme is permanently inactivated.

• Example: Acetylcholinesterase (AChE) Inhibitors:

  • Natural Function: AChE breaks down the neurotransmitter Acetylcholine (ACh) into Choline and Acetic Acid.

  • Organophosphates: These act as irreversible inhibitors of AChE.

  • Applications: They are used in insecticides and as chemical warfare agents (nerve agents).

  • Specific Nerve Agent: Novichok is cited as a notable example of a potent irreversible AChE inhibitor.

Allosteric Regulation and Kinetics

• Kinetic Curve Comparison:

  • Michaelis-Menten Enzymes: Exhibit a hyperbolic curve on a plot of reaction velocity ($V$) versus substrate concentration ($[S]$).

  • Allosteric Enzymes: Exhibit a sigmoidal (S-shaped) curve.

• Kinetics of Allosteric Enzymes:

  • They are usually multi-subunit proteins with several domains, regulatory chains, etc.

  • don’t follow Michaelis Menten behaviour

  • They exhibit co-operative substrate binding, combining two Michaelis Menten enzymes, one with a high value of Km another with low Km

  • The sigmoidal response is modelled as the combination of two states:

    1. T-state (Tense): Characterized by a high value of $K_m$ (lower affinity) not good at carrying out catalysis reactions.

    2. R-state (Relaxed): Characterized by a low value of $K_m$ (higher affinity) good at carrying out catalysis reactions.

• Allosteric Modulators:

  • Positive Allosteric Modulator (Allosteric Activator): Lowers [S] needed to reach half Vmax, shifts the curve to the left, decreasing the apparent $K_{0.5}$ (the allosteric equivalent of $K_m$) and making it easier to reach $V_{max}$.

  • Negative Allosteric Modulator (Allosteric Inhibitor): Increases [S] needed to reach half its Vmax, shifts the curve to the right, increasing the apparent $K_{0.5}$ and requiring higher substrate concentrations to achieve reaction velocity.

  • Modulators change the velocity without necessarily changing the absolute $V_{max}$, though they drastically change the activity at physiological substrate concentrations.

• Feedback Inhibition: A specific form of allosteric regulation where the end product of a metabolic pathway acts as an inhibitor for an enzyme acting earlier in the pathway (usually Enzyme 1). This prevents the over-accumulation of products.

  • cascade of signalling evens where several enzymes work on each others products to create whatever metabolites are needed for the metabolic pathway

  • end product of the metabolic pathway works as a negative modulator of some of the enzymes that are upstream in the metabolic pathway

  • already reached sufficiently high concentration of product therefore the pathway cant slow down

  • therefore the end product is a negative regulator of the enzymes at the beginning of the signalling cascade

Mechanisms for Regulation of Enzyme Activity

• Major Regulatory Strategies:

  1. Allosteric enzymes: Instantaneous control via non-covalent binding of modulators.

  2. Covalent modification: The chemical alteration of the enzyme by other enzymes.

  3. Induction or repression of enzyme synthesis: Regulating the actual amount of enzyme present in the cell.

Regulation by Covalent Modification

• Phosphorylation/Dephosphorylation:

  • Involves the reversible addition or removal of a phosphate group ($PO_4^{3-}$) onto a specific amino acid residue such as ser, thr, tyr, his, regulated by kinase and phosphatase regulatory enzymes. Can activate or deactivate the enzyme, enzyme specific

  • Kinases: Add phosphate groups to enzymes.

  • Phosphatases: Remove phosphate groups.

  • Residues involved: Phosphorylation typically occurs on the side chains of Serine (Ser), Threonine (Thr), Tyrosine (Tyr), and Histidine (His) residues.

• Activity Effects:

  • Activation: Some enzymes, such as MAPK kinase, become active when they are phosphorylated. causes catalytic site to be more active

  • Inactivation: Some enzymes, such as Glycogen synthase, become inactive when they are phosphorylated.

Regulation of Enzyme Synthesis

• Insulin Example:

  • The hormone insulin is a small protein produced in the pancreas.

  • High blood glucose levels stimulate an increase in insulin production.

  • Insulin increases the rate of synthesis (induction) of key enzymes involved in glucose metabolism, specifically:

    • Glucokinase

    • Phosphofructokinase

    • Pyruvate kinase

Summary of Enzyme Regulation Events