Enzymes and biocatalysis

Enzymes are proteins that act on substrates, catalyzing chemical reactions within the cell. Enzymes are specific in the sense that each enzyme only reacts with a few closely related substrates. Some enzymes require cofactors (biotin, lipoamide, cobalamin) to function properly. Enzymes can become denatured by changes in temperature or pH. Enzymes are classified as oxidoreductases, transferases, hydrolases, lyases, ligases, and isomerases, based on the type of reaction they catalyze. Enzyme kinetics is the study of enzyme reaction rates, which are determined using the Michaelis-Menten and Lineweaver-Burk equations. These equations can also be used to evaluate how different types of enzyme inhibitors affect the reaction rate. Enzymatic activity is precisely modulated through mechanisms such as allosteric regulation and covalent modification to maintain homeostatic control of metabolic pathways. Enzyme deficiencies can result in severe diseases such as Lesch-Nyhan syndrome, Gaucher disease, and phenylketonuria.

• Complex proteins that catalyze chemical reactions
• Act on substrates that can either be cleaved or joined to form a new product (e.g., carbonic anhydrase enzyme → CO₂ + H₂0 ⇄ H₂CO₃)
• Essential for life; if enzymes did not exist, cellular reactions would not occur fast enough to sustain life.
• Enzyme deficiencies can result in severe diseases (e.g., Lesch-Nyhan syndrome).
• Enzyme name is usually based on the reaction catalyzed plus the suffix “-ase”: e.g., for the enzyme that adds hydroxyl groups (OH^-) is formed as follows: hydroxyl + -ase → hydroxylase

General characteristics

• Apoenzyme: the inactive, protein-only component of an enzyme
• Cofactor: the non-protein "helper" molecule (organic or inorganic) that binds to the enzyme and is required for its activity
  • Inorganic cofactors: e.g., metal ions
  • Organic cofactors (coenzymes): e.g., B vitamins
• Holoenzyme: the complete, active enzyme, formed by the apoenzyme plus its cofactor
• Active site
  • Specific region on an enzyme where a particular substrate binds and where the enzymatic reaction occurs
  • Typically a pocket or cleft within the enzyme’s structure, tailored to fit the substrate precisely
• Regulatory site
  • Some enzymes have an additional binding site, which is involved in regulating enzyme activity.
  • Binding of specific molecules can either enhance or inhibit enzyme activity, allowing for fine-tuned control of metabolic pathways.
• Specificity
  • Enzymes are highly specific for their substrate and product (induced-fit model).
  • Some exceptions include proteases that break down proteins to peptides in the digestive system.
• Reaction rate: enzymes catalyze reactions by a factor of 10⁶–10¹¹
• Thermodynamics
  • Enzymes do not affect the energy level of substrates or products (free energy released remains the same).
  • Enzymes are able to decrease the energy of activation required to start a reaction.
  • The velocity of enzymatic reactions increases with temperature (up to 37^o C in humans).
• pH
  • Each enzyme has a specific pH at which it can achieve maximum velocity (V_max).
  • Alterations in pH can cause denaturation of enzymes (specific to each enzyme).
  • Example: Pepsin works best in acidic environment like the stomach (pH ∼1.5–2) and it is inactivated in the duodenum when bicarbonate is released from the pancreas, increasing the pH to > 7.

Spectrophotometry
Enzymatic activity (its reaction rate) is measured by monitoring product formation of the cofactors NAD(P)H and NAD(P)+. While both forms absorb light at 260 nm (due to their adenine base), only the reduced form, NAD(P)H, uniquely absorbs at 340 nm. Therefore, a reaction's rate can be quantified by measuring the change in absorbance at 340 nm over time. It is a direct assay, if the enzyme of interest directly consumes or produces NAD(P)H. If not, it is a coupled assay, where an indicator enzyme is added to link the primary reaction to the production or consumption of NAD(P)H.

Gibbs energy

Energy (∆G) for enzymatic reactions usually comes from the break down of ATP or GTP bonds (hydrolysis). Enzymatic reactions can occur spontaneously or nonspontaneously. The following are relationships between energy and enzymatic activity.

• Exergonic: Energy (∆G) < 0: Reactions can occur spontaneously (often irreversible).
• Endergonic: Energy (∆G) > 0: Reactions require energy to occur (from ATP or GDP).
• Balanced reaction: Energy (∆G) = 0: The reaction is at equilibrium (reversible).

Metal ions (inorganic cofactors)

• Definition: a coordinately (firmly) bound metal ion in the active site of an enzyme that participates in the catalytic reaction
• Function: can help stabilize structures, assist in electron transfer, or contribute to the formation of the product
• Examples
  • Zn²⁺ : e.g., in carbonic anhydrase
    • Mg²⁺ : e.g., in hexokinase, where it complexes with ATP
    • Fe²⁺/Fe³⁺: e.g., in cytochromes for electron transport
    • Cu²: e.g., in cytochrome c oxidase

Coenzymes (organic cofactors)

• Cosubstrates: loosely, temporarily bound
  • A type of cofactor that temporarily associates with the enzyme during the catalytic process but dissociates afterward; can be reused in different enzymatic reactions; e.g.,:
    • NAD⁺/NADH: carries electrons; derived from niacin
    • FAD/FADH₂: carries electrons; derived from riboflavin
    • Coenzyme A: carries acyl groups; derived from pantothenic acid
• Prostesthic groups: tightly, often covalently bound
  • E.g., biotin (carries CO₂ groups in carboxylation reactions), heme (binds O₂ in hemoglobin/myoglobin), thiamine pyrophosphate (TPP)

Overview of cofactors
Cofactor	Vitamin	Structure	Examples of enzymes	Reaction
Thiamine pyrophosphate (TPP)	Vitamin B1		Pyruvate dehydrogenase	Oxidative decarboxylation
FMN/FMNH2	Vitamin B2		Oxidoreductases	Electron transfer
FAD+/FADH2			Oxidoreductases	Electron transfer
NAD+/NADH	Vitamin B3		Oxidoreductases	Electron transfer
NADP+/NADPH			Oxidoreductases	Electron transfer
Coenzyme A	Vitamin B5		Citrate synthase	Acyl group transfer
Pyridoxal phosphate	Vitamin B6		Aspartate aminotransferase	Transamination Dehydration
Biotin	Vitamin B7		Carboxylases	Carboxyl group transfer (e.g., CO2)
Tetrahydrofolate	Vitamin B9		Methyltransferases	Methyl group transfer
Cobalamin	Vitamin B12		Methylmalonyl-CoA mutase	Alkyl group transfer
S-Adenosylmethionine (SAM)	N/A		Methyltransferases	Methyl group transfer
Lipoamide	N/A		Pyruvate dehydrogenase	Acyl group transfer Oxidative decarboxylation
Ascorbic acid	Vitamin C		Prolyl-4-hydroxylase	Electron transfer Hydroxylation
Phylloquinone	Vitamin K		Carboxylase	Electron transfer Carboxyl group transfer
Tetrahydrobiopterin	N/A		Hydroxylases	Electron transfer Oxygen atom transfer
ATP	N/A		Carboxylases	Phosphate group transfer

Michaelis-Menten kinetics

Description of an enzymatic reaction

• Enzymatic reactions with a hyperbolic curve (most common, e.g., alcohol dehydrogenase in ethanol oxidation): E + S ⇄ ES → E + P
  • [E] = enzyme
  • [S] = substrate
  • [P] = product
  • [V] = velocity
• A sigmoid curve indicates cooperativity (e.g., oxygen binding to hemoglobin)

Enzymatic reactions with a sigmoidal kinetic are indicative of cooperative binding (e.g., oxygen to hemoglobin).

Michaelis-Menten equation

• Equation: v = V_max [S] / (K_m + [S])
• Maximum velocity (Vmax)
  • Maximum rate at which an enzyme can catalyze a reaction
  • Directly proportional to the enzyme concentration
    • The only way to ↑ V_max is to increase [E] (cells achieve this, e.g., by increasing gene expression of a given enzyme)
    • ↑ Enzyme concentration → ↑ V_max
    • Noncompetitive inhibitors → ↓ [E] → ↓ V_max
• Michaelis constant: (K_m): the substrate concentration at which half of the active sites of the enzymes are bound to the substrate
  • Reaction velocity is ½ of V_max when the Michaelis constant concentration is reached
  • Inversely proportional to the affinity of the enzyme for the substrate: ↑ enzyme affinity → ↓ K_m
• Michaelis-Menten plot: a way of modeling Michaelis-Menten enzyme kinetics that relates the concentration of the substrate to reaction rate

Lineweaver-Burk equation and plot

Equation

• The Lineweaver-Burk equation is a double reciprocal of the Michaelis-Menten equation, where V = V_max [S] / K_m + [S] (if [E] remains constant), becomes 1 / v = K_m / V_max× 1/[S] + 1 / V_max.
• Represents enzyme kinetics in a linear graph rather than a hyperbola
• Equation is particularly important to determine the effect of drugs on enzymes

Plot

• Intercept with y-axis: : 1/V_max, the further from zero, the lower V_max
• Intercept with x-axis: : 1/-K_m : the closer to zero, the lower the affinity and the higher the K_m
• Slope: K_m/V_max

Very efficient and kompetent: On the Lineweaver-Burk plot, V_max usually represents the efficacy of a drug on the y-axis and K_m represents the potency on the x-axis.

To synchronize cellular processes and maintain homeostatic control, enzymatic activity must be precisely modulated rather than operating at constant maximum velocity. The cell employs diverse regulatory mechanisms such as allosteric modulation, covalent modification, and zymogen activation to upregulate, downregulate, or reversibly sequester enzymatic function in response to physiological demand.

Drug-response dynamics and enzyme inhibition

For details, see “Pharmacodynamics.”

Uncompetitive inhibitors are enzyme inhibitors that bind to the enzyme-substrate complex, decreasing K_m and V_max.

Kompetitive Inhibition: K_m Increases and V_max remains unchanged. Nonkompetitive Inhibition: No K_m change and V_max decreases.

Only Companions meet on the waY: Competitive inhibitors meet on the Y-axis (same V_max), noncompetitive do not.

Irreversible inhibition

• Definition: the permanent inactivation of an enzyme; activity can only be restored by synthesizing new enzyme molecules
• Mechanism: The inhibitor forms a strong covalent bond with the enzyme, often at the active site.
• Suicide substrates: a specific type of irreversible inhibitor (also called a mechanism-based inactivator); The enzyme's own active site converts the inhibitor into a reactive form, which then covalently bonds to and "kills" the enzyme.
  • Examples
    • Penicillin: acts as a suicide substrate for bacterial transpeptidase, an enzyme required for building the bacterial cell wall; this permanently blocks cell wall synthesis
    • Aspirin: irreversibly inhibits cyclooxygenase (COX) by covalently transferring an acetyl group to a serine residue in the active site, blocking prostaglandin synthesis

Allostery

Allostery is an important mechanism for altering the activity of an enzyme and thereby regulating metabolic pathways.

• Mechanism: A small molecule (an effector) binds reversibly to a specific allosteric site (the regulatory site) of an enzyme.
• Effect: causes a conformational change in the enzyme, altering the shape of the active site
  • Positive effector (activator): stabilizes the "R-state" (relaxed, high-affinity for substrate) conformation → increases enzyme activity
  • Negative effector (inhibitor): stabilizes the "T-state" (tense, low-affinity for substrate) conformation → inhibits enzyme activity
• Kinetics: Allosteric enzymes often have multiple subunits and display cooperative binding. This results in a sigmoidal (S-shaped) kinetics curve, not a hyperbolic one. This allows for a much more sensitive "on/off" switch in response to small changes in effector concentration.
• Example: Phosphofructokinase-1 (PFK-1) is the key rate-limiting step of glycolysis.
  • Inhibitors (negative): ATP, citrate (signal high energy)
  • Activators (positive): AMP, ADP, fructose-2,6-bisphosphate (signal low energy)

Allostery also occurs in proteins that are not enzymes (e.g., hemoglobin).

Feedback inhibition

A specific application of allosteric regulation used to control entire metabolic pathways

• Mechanism: The final end-product of a multi-step pathway acts as a negative allosteric effector for one of the first enzymes in that same pathway.
• Function: As soon as enough product is made, the product itself shuts down its own production line, preventing waste and resource depletion.

Covalent modification of enzymes

Enzymatic activity is also modulated through covalent modification, a process involving the stable attachment of chemical groups that functions as a molecular switch to rapidly alter metabolic flux in response to systemic signals.

• Phosphorylation/dephosphorylation: Kinases add a phosphate group (from ATP); phosphatases remove it.
  • Function: causes a major conformational change, toggling the enzyme between active and inactive states; primary control mechanism for cell signaling
  • Example: glycogen synthase and phosphorylase kinase (key enzymes in glycogen metabolism)
  • Phosphorylation cascades (one kinase activating many other kinases) are a major mechanism for signal amplification.
• Limited proteolysis (zymogen activation)
  • Mechanism: Enzymes are synthesized as inactive precursors called zymogens (or proenzymes). They are activated by the irreversible cleavage of a small peptide segment, which unmasks the active site.
  • Examples
    • Digestive enzymes: Trypsinogen (inactive, made in pancreas) is cleaved in the intestine to become trypsin (active). This prevents the pancreas from digesting itself.
    • Blood clotting: A complex cascade of zymogen activations (e.g., prothrombin → thrombin) leads to a rapid, massive response (a clot).
• ADP-ribosylation
  • Mechanism: transfer of an ADP-ribose group from NAD⁺ to a protein
  • Example: Cholera toxin uses this mechanism to irreversibly modify a G-protein, locking it in the "on" state. This causes massive ion and water secretion in the intestine, leading to severe diarrhea.

Regulation of enzyme concentration (gene expression)

This is the slowest, most long-term form of regulation.

• Mechanism: The cell controls the total amount of an enzyme by increasing (induction) or decreasing (repression) the transcription and translation of the gene that codes for it.
• Example: In response to high blood glucose, insulin induces the gene expression for enzymes like glucokinase and fatty acid synthase to store the excess fuel.

Compartmentation (spatial separation)

The cell uses its internal membranes (organelles) to segregate pathways, enzymes, and substrates.

• Mechanism: By confining an enzyme (e.g., in the mitochondrion) and its substrate (e.g., in the cytosol), the cell controls the reaction by controlling the transport of the substrate.
• Examples
  • Fatty acid breakdown (beta-oxidation) occurs in the mitochondria, while fatty acid synthesis occurs in the cytosol. This physical separation prevents the two opposing pathways from running simultaneously.
  • When glucose levels are low, a regulatory protein binds glucokinase and sequesters it in the nucleus, away from glucose in the cytosol. When glucose is high, the enzyme is released back into the cytosol to begin glycolysis.

• Glucose-6-phosphate dehydrogenase deficiency
• Chronic granulomatous disease
• Lesch-Nyhan syndrome
• Von Gierke's disease
• Pompe's disease
• Cori's disease
• Andersen's disease
• McArdle's disease
• Galactosemia
• Hereditary fructose intolerance
• Essential fructosuria
• Medium chain Acyl-CoA dehydrogenase deficiency
• Mucopolysaccharidoses
• Gaucher disease
• Krabbe disease
• Tay-Sachs disease
• Fabry disease
• Metachromatic leukodystrophy
• Niemann-Pick disease
• α1-antitrypsin deficiency
• Mitochondrial myopathies
• Alkaptonuria
• Homocystinuria
• Phenylketonuria
• Histidinemia