Enzyme Structure: Holoenzyme, Apoenzyme

• Prosthetic Group, Holoenzyme, and Apoenzyme are terms related to enzyme structure and function.
• Enzymes are biological catalysts that accelerate chemical reactions.

Prosthetic Group

• A prosthetic group is a non-protein molecule tightly and permanently bound to a protein (often an enzyme).
• It is vital for the protein's biological activity.
• Prosthetic groups can be organic (vitamins, sugars) or inorganic (metal ions).
• Heme, found in hemoglobin, catalase, and peroxidase, contains iron and is a prosthetic group.

Apoenzyme

• Apoenzyme is the protein part of an enzyme, lacking prosthetic groups or cofactors.
• Apoenzymes are usually inactive by themselves.
• Apoenzymes need prosthetic groups/cofactors to become active.

Holoenzyme

• Holoenzyme is the complete, catalytically active form of an enzyme.
• Holoenzymes have an apoenzyme (protein part) combined with its prosthetic group and any necessary cofactors.

Turnover Number (kcat)

• Turnover number (kcat) measures an enzyme's catalytic efficiency.
• kcat represents the maximum number of substrate molecules a single active site converts into product per unit time (usually per second).
• kcat is defined as Vmax/[E]T where Vmax is the max reaction rate when saturated with substrate and [E]T as the total enzyme concentration
• Higher kcat values indicate a more efficient enzyme, reflecting its intrinsic catalytic power.
• kcat is expressed in units of per second (s^-1).
• Catalase converts around 40 million molecules of H2O2 per second per active site.

Factors Affecting Turnover Number

• Enzyme structure (active site and overall conformation) influences kcat.
• At substrate saturation (when all active sites are occupied), kcat is at its maximum.
• Temperature and pH affect enzyme conformation and activity, thus influencing kcat.
• Inhibitors can lower kcat by binding to the enzyme and reducing its activity.

Specific Activity

• Specific activity assesses the purity of an enzyme preparation.
• It quantifies enzyme catalytic activity per unit of total protein.
• Specific activity is the number of enzyme units per milligram of total protein.
• One enzyme unit converts one micromole of substrate per minute under specific conditions.
• Higher specific activity indicates a purer enzyme preparation and should increase during purification as contaminants are removed.

Calculation

• Specific activity is enzyme activity divided by total protein concentration.
• Enzyme activity is measured in enzyme units (e.g., µmol/min).
• Total protein concentration is measured in milligrams per milliliter (mg/mL).
• An enzyme preparation with an activity of 100 µmol/min and a total protein concentration of 2 mg/mL has a specific activity of 50 units/mg.

Katal (kat)

• Katal (kat) is the SI unit of catalytic activity of an enzyme.
• One katal converts one mole of substrate per second under specified conditions.
• Katal is internationally recognized for expressing enzyme activity.
• One katal represents a significant amount of enzyme activity, so nanokatal (nkat) or picokatal (pkat) are more commonly used.
• 1 katal = 60 micromoles per minute (µmol/min).
• 1 katal = 6 x 10^7 units (where 1 unit converts 1 micromole of substrate per minute).
• An enzyme sample with an activity of 10 nanokatals (nkat) converts 10 nanomoles of substrate into product per second under specific conditions.

IU (International Unit)

• The International Unit (IU) is a unit of measurement for the catalytic activity of enzymes.
• 1 IU of an enzyme converts one micromole (μmol) of substrate per minute under specific conditions.
• The IU is used in biochemistry, clinical chemistry, and pharmacology.
• Specific conditions significantly influence the enzyme's activity.
• 1 IU is equivalent to 1/60 µkat (microkatal).
• An enzyme preparation with an activity of 100 IU converts 100 μmol of substrate to product per minute under specific conditions.

The IUB/EC Classification

• The IUB/EC classification system categorizes enzymes based on the type of chemical reaction they catalyze.
• There are six main classes

Six Main Classes

• Oxidoreductases (EC 1) catalyze oxidation-reduction reactions (electron transfer), examples are dehydrogenases and oxidases.
• Transferases (EC 2) catalyze the transfer of functional groups between molecules, examples are kinases.
• Hydrolases (EC 3) catalyze the hydrolysis of various bonds, examples are proteases and lipases.
• Lyases (EC 4) catalyze the addition or removal of groups to or from a double bond,or the formation of a double bond by removal of a group, examples are aldolases and decarboxylases.
• Isomerases (EC 5) catalyze the interconversion of isomers (same chemical formula, different structures), examples are racemases and epimerases.
• Ligases (EC 6) catalyze the joining of two molecules, often coupled with ATP hydrolysis, examples are synthetases.

Activation Energy

• Activation energy is the minimum energy required for a chemical reaction to occur.
• Activation energy can be likened to a hill that that reactants need to climb before transforming into products.
• Activation energy represents the energy barrier that separates reactants from products.
• Reactants must reach the transition state (activated complex), which is an unstable intermediate state.
• Reactions with lower activation energies have faster reaction rates.
• Catalysts lower activation energy, making reactions occur more readily.
• Higher temperatures provide more energy to reactants, increasing the likelihood of overcoming the activation energy barrier.
• Some molecules are inherently more reactive than others.
• Catalysts provide an alternative reaction pathway with a lower activation energy.

Mechanism of Enzyme Action:

• Enzymes speed up chemical reactions in living organisms.

Active Site

• The active site is a specific region on the enzyme where the substrate binds, usually a small pocket of few amino acids.
• The active site's shape and chemical properties determine substrate specificity.

Enzyme-Substrate Complex

• The enzyme and substrate interact to form an enzyme-substrate complex, which is typically temporary and reversible.
• Hydrogen bonds, ionic interactions, and hydrophobic interactions hold the complex together.

Catalysis

• Once the substrate is bound, the enzyme facilitates the chemical reaction by lowering activation energy.
• Enzymes offer a different route for the reaction with a lower activation energy by orienting substrates or stabilizing the transition state.

Product Release

• After the reaction, the products are released from the enzyme's active site.
• The enzyme returns to its original state and can bind another substrate molecule.

Single-Substrate Reactions

• In single-substrate reactions, one substrate molecule binds to the enzyme's active site and is transformed into a product.
• Lactase breaks down lactose (a single substrate) into glucose and galactose (products).

Bi-Substrate Reactions

• Bi-substrate reactions involve two different substrate molecules binding to the enzyme's active site simultaneously.
• The enzyme facilitates the reaction between these two substrates.
• Hexokinase catalyzes the reaction between glucose and ATP (two substrates) to form glucose-6-phosphate and ADP (products).

Factors Affecting Enzyme Activity

• Enzymes have an optimal temperature range; high temperatures can denature the enzyme.
• Enzymes have an optimal pH range; changes in pH can affect the enzyme's structure.
• Increasing substrate concentration generally increases enzyme activity up to saturation.
• Inhibitors bind to the enzyme and reduce its activity.
• Some enzymes require cofactors (non-protein molecules) for activity.

Enzyme Inhibitors

• Enzyme inhibitors are molecules that bind to enzymes, reducing their catalytic activity and slowing down or stopping biochemical reactions.

Types of Enzyme Inhibitors

• Reversible inhibitors bind through weak interactions (hydrogen bonds, van der Waals forces), and their inhibition can be overcome.
• Competitive inhibition involves the inhibitor having a similar shape to the substrate and competes with the substrate for binding to the enzyme's active site. An example is malonate inhibiting succinate dehydrogenase.
• Non-competitive inhibition involves the inhibitor binding to a site different from the active site (allosteric site), changing the enzyme's shape and preventing substrate binding. An example is cyanide inhibiting cytochrome c oxidase.
• Uncompetitive inhibition involves the inhibitor binding only to the enzyme-substrate complex, preventing product formation. Some antibiotics act this way.
• Irreversible inhibitors bind to enzymes through strong, covalent bonds, and the inhibition is permanent.
• Organophosphate pesticides irreversibly inhibit acetylcholinesterase, an enzyme crucial for nerve function.

Significance of Enzyme Inhibitors

• Many antiviral and anticancer drugs target specific enzymes in pathogens or cancer cells.
• Many pesticides act as enzyme inhibitors in insects, disrupting their metabolic processes.
• Enzyme inhibitors are used as research tools to study enzyme mechanisms and metabolic pathways.