Enzymes and biocatalysis

Overview

Enzymes are complex proteins that catalyze chemical reactions. Enzymes act on substrates that can either be cleaved or joined to form a new product (e.g., carbonic anhydrase enzyme → CO2 + H20 ⇄ H2CO3). They are essential for life, if enzymes did not exist, cellular reactions would not occur fast enough to sustain life. Thus, enzyme deficiencies can result in severe diseases (e.g., Lesch-Nyhan syndrome).

The name of enzymes is usually based on the reaction catalyzed plus the suffix -ase. For example, the name of the enzyme that adds hydroxyl groups (OH-) formed as follows: hydroxyl + -asehydroxylase.

General characteristics of enzymes

  • Active site: binding site for a specific substrate on a specific enzyme
  • Specificity: Enzymes are highly specific for their substrate and product.
  • Rate: enzymes catalyze reactions by a factor of 106–1011
  • Coenzymes: many enzymes require coenzymes (e.g., biotin) that allow them to perform their action on a substrate.
    • Usually small organic molecules derived from metal ions or vitamins
  • 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 37o C in humans).
      • Temperatures > 37o C slow down enzymatic reactions and can result in denaturation of enzymes.
  • pH: Each enzyme has a specific pH at which it can achieve maximum velocity (Vmax).

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)

Classes of enzymes

Enzyme class Function Subclass Examples
Oxidoreductases
  • Dehydrogenases
  • Oxidases
  • Oxygenases
  • Hydroxylases
    • Transfers hydroxyl groups (OH)
Transferases
  • Transfer functional groups
  • Kinases
    • Transfer phosphate groups from a high energy molecule (e.g., ATP, ADP) onto a substrate
  • Phosphorylases
    • Add inorganic phosphate onto substrates
    • Do not require any energy source
  • Aminotransferases
  • Glycosyltransferases
Hydrolases
  • Cleave covalent bonds by adding water
Lyases
  • Aldolases
  • Decarboxylases
  • Dehydratases
Isomerases
  • Converts a substrate into its isomer
  • Mutases
    • Move functional groups within a molecule
  • Epimerases
Ligases

Energy carriers

Base molecule Transferred group Carrier of energy Released energy Metabolic site
ADP Phosphate ATP -31 KJ/mol
  • Ubiquitous energy source
GDP Phosphate GTP

-31 KJ/mol

  • TCA
Creatine Phosphate PKr

-43 KJ/mol

CoA Thioester Acetyl-CoA

-36 KJ/mol

  • TCA
Pyruvate Phosphate PEP

-62 KJ/mol

Cofactors

Cofactor Vitamin Structure Reaction
Thiamine pyrophosphate (TPP) Vitamin B1 Oxidative decarboxylation
FMN/FMNH2 Vitamin B2 Electron transfer
FAD+/FADH2 Vitamin B2 Electron transfer
NAD+/NADH Vitamin B3 Electron transfer
NADP+/NADPH Vitamin B3 Electron transfer
Coenzyme A Vitamin B5 Acyl group transfer
Pyridoxal phosphate Vitamin B6 Transamination, dehydration
Biotin Vitamin B7 Carboxyl group transfer
Tetrahydrofolate Vitamin B9 Methyl group transfer
Cobalamin Vitamin B12 Alkyl group transfer
S-Adenosylmethionine (SAM) Methyl group transfer
Lipoamide Oxidative decarboxylation
Ascorbic acid Vitamin C

Electron transfer and hydroxylation

Phylloquinone Vitamin K Electron and carboxyl group transfer
Tetrahydrobiopterin

Electron and oxygen atom transfer

ATP Phosphate group transfer

For more information, see vitamins.

Rate-limiting enzymes

Pathway Enzyme Stimulation Inhibition
Glycolysis
  • Fructose-2,6-biphosphate
  • AMP
Gluconeogenesis
  • Fructose-1,6-biphosphatase
  • Fructose-2,6-biphosphate
  • AMP

Citric acid cycle

  • ADP
  • Ca2+
Glycogenesis
  • Dephosphorylation upon insulin signaling
  • Glucose-6-phosphate

Glycogenolysis

  • Dephosphorylation upon insulin signaling
  • ATP
  • Glucose-6-phosphate

Pentose phosphate pathway

(HMP shunt)

Pyrimidine synthesis
  • UTP
Purine synthesis
  • ADP, ATP
  • GDP, GTP
Urea cycle
Fatty acid synthesis

β-oxidation

  • Carnitine acyltransferase I
  • Malonyl-CoA
Cholesterol synthesis
  • HMG-CoA reductase

Enzyme kinetics

Michaelis-Menten kinetics

  • [E] = enzyme, [S] = substrate, [P] = product, [V] = velocity
    • E + S ⇄ ES → E + P
  • Maximum velocity (Vmax): maximum rate at which an enzyme can catalyze a reaction
  • Michaelis constant: (Km): the substrate concentration at which half of the active sites of the enzymes are bound to the substrate
    • Reaction velocity is ½ of Vmax when the Michaelis constant concentration is reached
    • Inversely related to the affinity of the enzyme for the substrate
  • Michaelis-Menten equation: v = Vmax [S] / (Km + [S])

Km Vmax
  • Inversely proportional to the affinity of the enzyme for the substrate
    • ↑ Enzyme affinity → ↓ Km
  • Directly proportional to the enzyme concentration
    • ↑ Enzyme concentration → Vmax
  • The only way to Vmax is to increase [E]
    • Cells achieve this, e.g., by increasing gene expression of a given enzyme
  • Noncompetitive inhibitors → ↓ [E] → Vmax

Lineweaver-Burk equation and plot

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

  • Intercept with y-axis: 1/Vmax: the further from zero, the lower Vmax
  • Intercept with x-axis: 1/-Km : the closer to zero, the lower the affinity
  • Slope: Km/Vmax

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

Drug-response dynamics

The details of pharmacodynamics are explained in the learning card on the fundamentals of pharmacology.

Parameter Uncompetitive inhibitors

Noncompetitive inhibitors

Competitive inhibitors (reversible)

Competitive inhibitors (irreversible)

Similar to the substrate
  • No
  • No
  • Yes
  • Yes
Effect of increased [S]
  • None
  • None
  • Inhibition can be overcome
  • None
Binding site
  • Binds to enzyme-substrate complex
Effect on Km
  • Decreased
  • None
  • Increased
  • None
Effect on Vmax
  • Decreased
  • Decreased
  • None
  • Decreased
Pharmacodynamic effect
  • ↓ Enzyme efficacy
  • ↓ Enzyme efficacy
  • Does not affect enzyme efficacy
  • Potency

For more information on the effects of inhibitors and pharmacodynamics, see types of drug-receptor interactions.

Uncompetitive inhibitors are enzyme inhibitors that bind to the enzyme-substrate complex, decreasing Km and Vmax.

Kompetitive Inhibition: Km Increases; Vmax unchanged. Nonkompetitive Inhibition: No Km change; Vmax decreased.

Clinical significance

  • Kaplan. USMLE Step 1 Lecture Notes 2016: Pharmacology. New York, NY: Kaplan; 2015.
  • Kaplan. USMLE Step 1 Lecture Notes 2018: Biochemistry and Medical Genetics. New York, NY: Kaplan; 2017.
  • Le T, Bhushan V,‎ Sochat M, Chavda Y, Abrams J, Kalani M, Kallianos K, Vaidyanathan V. First Aid for the USMLE Step 1 2019. New York, NY: McGraw-Hill Medical.
last updated 11/28/2019
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