Enzymes 2: Metabolic Control & Pharmacological Inhibition

Enzymes 2: Metabolic Control & Pharmacological Inhibition

Case Study: Atrial Fibrillation, Hypercholesteremia, and Angina

• An 80-year-old male with atrial fibrillation, hypercholesterolemia, and angina was taking multiple medications.

  • Warfarin (5 mg OD) for atrial fibrillation: An anticoagulant that prevents blood clots.

  • Simvastatin (20 mg) and gemfibrozil for hypercholesterolemia: Simvastatin is an HMG-CoA reductase inhibitor, reducing cholesterol synthesis, while gemfibrozil is a fibric acid derivative that lowers triglyceride levels and increases HDL cholesterol.

  • Isosorbide mononitrate and sublingual nitroglycerin for angina: These are nitrates used to dilate blood vessels and improve blood flow to the heart, reducing chest pain.

• Referred to an oral surgeon for premolar tooth pain and hyperplastic tissue in the right maxillary first and second molar region.

  • The oral surgeon noted hyphae-like structure and redness, suspecting a fungal infection and prescribed fluconazole (200 mg OD for 14 days).

• Fluconazole:

  • Inhibits fungal cytochrome P-450 sterol C-14 α-demethylation: This enzyme is essential for the synthesis of ergosterol, a key component of fungal cell membranes.

  • Prevents conversion of lanosterol to ergosterol.

  • Inhibits membrane sterol synthesis.

  • Prevents fungal replication.

Complications and Drug Interaction

• Cardiologist managed the warfarin regime, while the family care physician managed other medications.

• Fluconazole was prescribed by a pharmacy not fully aware of all the patient's medications.

  • A drug interaction report was sent to the primary physician but not reviewed until day 9 of fluconazole treatment.

• A day later, the patient exhibited confusion and drove erratically, leading to a suspected stroke.

  • Hospitalization revealed a significant right frontal lobe bleed.

  • The patient's condition deteriorated, and eventually, they died.

• Fluconazole:

  • Competitively inhibits human liver cytochromes (e.g., CYP2C19): CYP2C19 is involved in the metabolism of many drugs, including warfarin.

  • Inhibits warfarin's metabolism, leading to excessive blood levels.

  • Can lead to major bleed events and stroke.

Learning Objectives

• Understand enzyme kinetics via the Michaelis-Menten equation.

• Categorize enzymatic inhibitors and infer their mode of action based on changes in kinetic parameters.

• Explore multi-subunit enzymes and the nature of cooperativity.

• Explain different modes of enzyme regulation.

Describing Enzyme Kinetics

• In the simplest enzyme-catalyzed reactions:

  • $E + S \rightleftharpoons [ES] \rightarrow E + P$

  • $k<em>1, k</em>{-1}, k<em>2 = k</em>{cat}$

Michaelis-Menten Kinetics

• Plotting $v_0$ against $[S]$ with a fixed $[enzyme]$ yields a hyperbolic curve for most enzymes.

• Michaelis-Menten equation:

  • $v<em>0 = \frac{V</em>{max} [S]}{K_m + [S]}$

  • $V[max]$ is the maximum possible rate at that $[enzyme]$ . It represents the theoretical maximal rate of the reaction when the enzyme is fully saturated with the substrate.

  • $K<em>m$ is the substrate concentration resulting in $V[max]/2$. It is a measure of the affinity of the enzyme for its substrate. A low $K</em>m$ indicates high affinity, meaning only a small amount of substrate is needed to achieve $V[max]/2$.

Vmax and Km

• $V[max]$:

  • Theoretical rate at an infinitely high substrate concentration.

  • All enzyme molecules contain bound substrate.

  • Directly proportional to the amount of enzyme.

• $K_m$ (Michaelis constant):

  • Substrate concentration at which the initial rate is $1/2 V[max]$.

  • Changes in $[S]$ close to $K_m$ have a large effect on velocity.

  • $[S]$ to saturate enzyme can be calculated if $K_m$ is known.

  • High $K_m$ = low substrate efficacy (and vice-versa).

Comparing Isozymes

• Hexokinase isozymes are found in red blood cells and liver.

• Glucokinase (liver) has a $K_m$ of ~10 mM and a high $V[max]$: Allows the liver to effectively clear glucose from the blood after a meal.

• Hexokinase I (RBCs) has a $K_m$ of 0.05 mM and a low $V[max]$: Ensures that red blood cells can still perform glycolysis even when glucose levels are low.

• Hexokinase I permits glycolysis to proceed even with very low blood glucose levels.

• Glucokinase directs glucose to storage as glycogen, but only when it is in excess of energy needs.

Enzyme Inhibitors

• Bind to the enzyme and block catalysis either directly or indirectly.

  • Competitive (direct inhibition): Binds to the active site, preventing substrate binding.

  • Non-competitive or uncompetitive (indirect): Binds elsewhere, altering enzyme shape or function.

• Effects from binding outside the active site are termed allosteric.

Characterizing Enzyme Inhibitors

• Characterized by the inhibition constant $K_i$ (where binding is reversible).

  • Equivalent of $K_d$ for ligand binding: Represents the dissociation constant.

  • Smaller values (5 nM vs 5 μM) represent higher affinity.

• Classifying inhibitors is useful to:

  • Give insights into catalytic mechanisms and metabolic control.

  • Help characterize inhibitors as therapeutic agents.

Kinetics of Competitive Inhibitors

• Competes directly with substrate at the substrate-recognition site.

• Can be overcome by increasing $[substrate]$.

  • Apparent $K_m$ is increased, while $V[max]$ is unchanged: Because more substrate is needed to achieve $V[max]/2$ in the presence of the inhibitor.

Non-Competitive Inhibition

• If an inhibitor does not compete with a substrate for its binding site:

  • Termed non-competitive inhibition.

  • Uncompetitive inhibition is a special case where the inhibitor only binds to the transition-state conformation.

• Non-competitive inhibitors lower $V[max]$ as they reduce the concentration of active enzyme.

• $K_m$ is unchanged (substrate binding is unaffected).

Kinetics of Non-Competitive Inhibition

• $V[max] \downarrow$: Effect of inhibitor cannot be reversed by $\uparrow [S]$.

• $K_m$ unchanged: Inhibitors do not interfere with substrate binding.

Kinetics of Other Types of Inhibition

• It is rare for inhibitors to bind to enzymes without affecting the affinity for the substrate.

• Other forms of inhibition include:

  • Mixed inhibition: Both $K_m$ and $V[max]$ change with inhibitor bound (forms EI and ESI complex as for non-competitive but binds with different affinities).

  • Uncompetitive inhibition: Both $K_m$ and $V[max]$ fall (forms dead-end ESI complex).

  • Substrate and product inhibition: Allosteric modulation since substrates and/or products are binding away from the active site.

  • Irreversible inhibition: Special case where kinetic analyses don’t apply.

Cooperativity

• When a substrate binds on a subunit in a complex with multiple catalytic sites, it can affect catalysis at the other subunits:

  • Positive cooperativity: catalysis gets faster as more substrate binds (sigmoidal curve).

  • Negative cooperativity: catalytic activity becomes slower than expected.

"Concerted" Cooperativity Model

• Enzyme subunits adopt two conformations: T (taut) & R (relaxed).

• Subunits all start in the low-affinity “T” conformation, which has a weak affinity for the substrate.

• Binding of the first substrate molecule stabilizes the high affinity “R” state for all subunits.

• Activators increase the fraction of enzyme in the R state.

The “Sequential” Model

• Binding of each substrate molecule incrementally increases the stability of the “R” state.

• This makes binding to remaining subunits progressively easier.

• Can explain negative cooperativity (inhibitors stabilize the T-state).

Enzyme Regulation: An Overview

• Enzymes must be regulated to maintain metabolic effectiveness.

Enzyme Regulation (2)

• The slowest (“rate-limiting”) step of a metabolic pathway is the most efficient control point.

• Longer divergent pathways are controlled at the steps after forks.

• Regulatory mechanisms include:

  • Modulator binding (allosteric modulation).

  • Phosphorylation (& other covalent modifications).

  • Protein-protein interactions.

  • Proteolytic cleavage.

Mechanisms of Enzyme Regulation

• Enzyme Binding

  • Allosteric modulators.

  • Protein interactions.

• Control of protein levels

  • Degradation.

  • Synthesis.

• Covalent modification

  • Phosphorylation & dephosphorylation.

  • Cleavage.

Allostery: A Definition

• Allostery involves a change in protein conformation following binding at a site distinct from the natural binding site.

• In allosteric regulation, binding affects the activity of the target protein (e.g., catalytic activity for enzymes).

• Binding at allosteric sites can either inhibit or activate.

• With the exception of competitive inhibition, enzyme inhibitors act allosterically.

Allosteric Regulation

• Regulation is through reversible binding of small molecules (modulators or effectors).

• Effectors bind at allosteric sites (not the active/binding site).

• Their binding changes the active-site conformation (can potentiate or inhibit).

• Allosteric regulation is fast, so it is often the first response of cells to changes in conditions.

Allosteric Regulation & Multimers

• Allosterically-regulated enzymes are often composed of multiple subunits.

• The allosteric site is often not on the catalytic subunit.

• Where there is more than one active site, allosteric modulators will usually exhibit sigmoid kinetics.

Kinetics of Multimer Regulation

• Allosteric effectors are associated with sigmoidal kinetics when they bind to cooperative (multimeric) enzymes.

• $K*{0.5}$ ($[substrate]$ to give 50% enzyme saturation) used instead of $K_m$.

• Activators & inhibitors can affect $K*{0.5}$ and/or $V[max]$.

Advantages of Allosteric Regulation

• Enzymes can be inhibited or activated.

• No similarity to substrate or product is required.

• Regulation is rapid.

• Effectors that increase enzyme activity are allosteric activators/positive effectors.

• Effectors that decrease enzyme activity are allosteric inhibitors/negative effectors.

Allosteric Regulation: Examples

• Many key enzymes in metabolic oxidation pathways are regulated allosterically.

• Examples:

  • Phosphofructokinase (glycolysis): Activated by AMP and ADP, inhibited by ATP and citrate.

  • Isocitrate dehydrogenase (TCA cycle): Activated by ADP, inhibited by ATP and NADH.

• Regulation is often by ADP or AMP.

• Example: Muscle glycogen phosphorylase degrades glycogen to glucose-1P.

  • AMP is an allosteric activator (levels increase as ATP is used).

  • The same enzyme is also regulated by covalent modification.

Regulation: Covalent Modification

• Covalent modifications can lead to conformational changes.

• Phosphorylation

  • Addition of phosphate to serine (S), threonine (T), or tyrosine (Y).

  • $PO_4^{3-}$ is a bulky, negatively charged group.

  • Interacts strongly with nearby residues and alters ionic interactions and H-bonding.

  • Mediated by protein kinases (add phosphates using ATP) & phosphatases (remove them by hydrolysis).

Protein Kinases

• Some protein kinases only regulate one protein, while others regulate several.

• Example: Protein Kinase A (PKA), a serine/threonine kinase:

  • Phosphorylates several enzymes across a number of different metabolic pathways.

  • PKA is activated by the second messenger 3’,5’-cyclic AMP (cAMP) binding to regulatory subunits, which then dissociate.

  • Several receptors affect $[cAMP]$ (e.g., β-adrenoceptors, dopamine D1 receptors).

Regulation: Protein-Protein Interactions

• Interactions between proteins can change their conformation, affecting the active site.

• Example: The calcium-calmodulin family:

  • Modulate activity of many target enzymes through steric hindrance.

  • Activated by $Ca^{2+}$ binding.

  • Example: Glycogen phosphorylase kinase is inhibited by bound calmodulin:

    • $\uparrow [Ca^{2+}]$ signals muscle contraction.

    • $Ca^{2+}$-calmodulin dissociates from the GP-kinase, activating the enzyme.

Regulation: Proteolytic Cleavage

• Enzymes can be irreversibly activated (e.g., clotting cascade) or inactivated (e.g., HMG-CoA reductase) by proteolytic enzymes.

• The overall enzyme activity in a cell is controlled by degradation of enzymes by intracellular proteases in lysosomes or proteasomes.

Regulation: Enzyme Synthesis

• Enzymes' activity can also be controlled at the point of synthesis.

• Induction or repression leads to an alteration in the total population of active sites.

• Generally, such control is limited to those enzymes required at specific points in development or under particular physiological conditions.

Summary

• The kinetic properties of simple enzymes can be described by the Michaelis-Menten (M-M) equation and the parameters $V[max]$ and $K_m$.

• Inhibitors affect enzyme kinetics in different ways depending on how and where they bind (competitive, non-competitive, etc.).

• More complex enzymes cannot be described by M-M kinetics, including those that exhibit cooperativity (interaction between multiple binding sites).

• Enzymes are highly regulated and can be controlled by ligand binding, covalent modification, and by synthesis/degradation.

Michaelis-Menten Caveats

• Michaelis-Menten kinetics only work for relatively simple enzymes and assume:

  • A single substrate reaction where the enzyme-substrate complex (ES) can freely dissociate.

  • A single independent active site (i.e., not a multi-enzyme complex).

  • $[substrate] >> [enzyme]$; therefore, $[S] >> [ES]$.

• These caveats notwithstanding, Michaelis-Menten kinetics are a useful way to compare enzymes.

Uncompetitive Inhibition

• An uncompetitive inhibitor can only bind to the enzyme-substrate complex (ES), and not to the free enzyme.

• The inhibitor must therefore bind to a site created by conformational change (or to the substrate).

• Creates a dead-end complex (ESI).

• Cannot be overcome by increasing $[S]$.

• $V[max]$ is therefore reduced (less product formation).

• $K_m$ is also reduced (apparent affinity of substrate for enzyme increased).

Mixed-reversible Inhibition

• Where $k<em>{i1} ≠ k</em>{i2}$, then:

  • Both $K_m$ and $V[max]$ change with the inhibitor present.

  • This is termed mixed inhibition.

• In the Lineweaver-Burk plot, the lines for inhibited vs. uninhibited can cross above or below the x-axis.

Effects of Chemical Modifications

• Phosphorylated enzymes may be more or less active than their unphosphorylated forms.

• Examples:

  • Phosphorylation of glycogen synthase decreases its activity.

  • Glycogen phosphorylase activity is increased by phosphorylation.

• Other covalent modifications include the addition/removal of acetyl, ADP-ribose, and lipid moieties.

• These can affect the ability of the enzyme to interact with other proteins or may change its localization.

Trimeric G-Proteins

• Trimeric G Proteins:

  • Transduce signals from G-protein-coupled receptors.

  • Protein interactions control their activation (via GDP-GTP exchange and a and bg subunit dissociation).

  • G protein a-subunits are GTPases, so they inactivate over time.

  • All pathway components can also be regulated allosterically.

Telangiectasia-ectodermal dysplasia-brachydactyly-cardiac anomaly syndrome

• Believed to have been caused by de novo mutations in Protein Kinase D1

  • Mutation c.1774G>C, p.(Gly592Arg) in exon 12 of the PRKD1 gene

  • Serine/threonine