06 Enzymes

What are the general properties of enzymes?

Enzymes are proteins (except some RNA ribozymes), highly specific, efficient, reusable, and catalyze reactions without being consumed.

Why are enzymes considered biological catalysts?

They accelerate biological reactions by lowering activation energy but do not change equilibrium or overall free energy.

How do enzymes compare to inorganic catalysts?

Enzymes work under mild physiological conditions (pH ~7, temp ~37°C) and show much higher specificity and efficiency.

What is catalytic power?

The ability of enzymes to increase the rate of reactions by factors of 10^7–10^16 compared to uncatalyzed rates.

What is specificity in enzymes?

The ability of an enzyme to choose exact substrates and reactions among many possibilities.

Why is enzyme specificity important biologically?

It ensures correct metabolic pathways proceed without harmful side reactions.

What are the two main systems of enzyme nomenclature?

Common/trivial names (e.g., trypsin) and systematic names assigned by the Enzyme Commission (EC).

What are the seven major classes of enzymes?

Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, Ligases, Translocases.

What reactions do oxidoreductases catalyze?

Oxidation-reduction reactions, involving electron transfer often with NAD⁺, NADP⁺, FAD, or FMN.

Give two examples of oxidoreductases.

Dehydrogenases and oxidases.

What reactions do transferases catalyze?

Transfer of functional groups like amino, methyl, acyl, phosphoryl, glycosyl.

Give two examples of transferases.

Kinases (transfer phosphate) and aminotransferases (transfer amino groups).

What reactions do hydrolases catalyze?

Bond cleavage by hydrolysis, using water as reactant.

Give two examples of hydrolases.

Esterases and peptidases.

What reactions do lyases catalyze?

Non-hydrolytic cleavage of C–C, C–N, C–S bonds, often forming double bonds.

Give two examples of lyases.

Aldolase and decarboxylases.

What reactions do isomerases catalyze?

Intramolecular rearrangements (e.g., racemization, epimerization, mutase reactions).

Give two examples of isomerases.

Phosphoglucose isomerase, triose phosphate isomerase.

What reactions do ligases catalyze?

Formation of new covalent bonds (C–C, C–O, C–N, C–S) coupled to ATP hydrolysis.

Give two examples of ligases.

DNA ligase and pyruvate carboxylase.

What reactions do translocases catalyze?

Transport of molecules or ions across membranes or within membranes.

Give an example of a translocase.

ATP synthase (proton translocase).

What does the first number in an EC classification mean?

It indicates the enzyme class (e.g., 1 = oxidoreductases).

What is an apoenzyme?

The inactive protein portion of an enzyme lacking its cofactor.

What is a holoenzyme?

The active enzyme consisting of apoenzyme plus its required cofactor.

What is a prosthetic group?

A tightly bound non-protein component essential for enzyme activity (often a metal ion or organic group).

What is a coenzyme?

A small organic molecule that acts as a transient carrier of specific atoms or groups in catalysis.

What is a cofactor?

A non-protein substance (metal ion or organic molecule) required for enzyme activity.

What are metalloenzymes?

Enzymes with metal ions tightly bound as prosthetic groups.

What are metal-activated enzymes?

Enzymes that bind metal ions loosely and reversibly.

Give three examples of metalloenzymes.

Cytochrome oxidase (Cu, Fe), carbonic anhydrase (Zn), catalase (Fe).

Give three examples of metal-activated enzymes.

Kinases (Mg²⁺), amylases (Ca²⁺), DNA polymerase (Mg²⁺).

What is the active site?

The small region of the enzyme where substrate binds and catalysis occurs.

What are the main features of the active site?

Small portion of enzyme, specific shape, complementary to substrate, contains catalytic residues, binds via weak interactions.

What is substrate binding usually mediated by?

Hydrogen bonds, hydrophobic interactions, ionic bonds, and van der Waals forces.

What are the catalytic residues in serine proteases?

Asp102, His57, Ser195.

What is stereochemical specificity?

Enzyme distinguishes between stereoisomers and acts on only one.

What is group specificity?

Enzyme recognizes a functional group rather than the whole molecule.

What is linkage specificity?

Enzyme recognizes a particular type of bond independent of the rest of the structure.

What is absolute specificity?

Enzyme acts only on one substrate for one reaction.

What is the lock-and-key model?

Substrate fits exactly into a rigid active site like a key into a lock.

What is the induced-fit model?

Substrate binding induces conformational change in the enzyme to optimize interaction.

What is proximity effect in enzyme catalysis?

Enzymes bring substrates close together to increase reaction rate.

What is orientation effect in enzyme catalysis?

Enzymes orient substrates in the correct alignment for reaction.

What is entropy reduction in enzyme action?

Enzyme binding reduces random motion (translational/rotational entropy), making reaction more likely.

What are two types of entropy relevant to enzyme catalysis?

Translational entropy and rotational entropy.

What is structural strain in enzyme catalysis?

Enzyme distorts substrate into a geometry resembling the transition state, facilitating bond breakage.

What is desolvation in enzyme catalysis?

Exclusion of water molecules upon substrate binding, strengthening enzyme-substrate interactions.

What is electrostatic destabilization in enzyme catalysis?

Introduction of repulsive charges that drive substrate toward reaction.

What is preferential binding of the transition state?

Enzymes bind transition states more tightly than substrates, lowering activation energy.

What is general acid catalysis?

Enzyme donates a proton to stabilize negative charges in transition state.

What is general base catalysis?

Enzyme accepts a proton to stabilize positive charges or activate nucleophiles.

What is covalent catalysis?

Formation of a transient covalent bond between enzyme and substrate.

Give examples of nucleophilic side chains involved in covalent catalysis.

Serine OH, cysteine SH, lysine NH₂, histidine imidazole.

What is metal ion catalysis?

Enzyme uses bound metal ions to stabilize charges, participate in redox, or assist in substrate binding.

Give three roles of metal ions in catalysis.

Orient substrate, mediate redox, shield/stabilize negative charges.

How does Mg²⁺ assist ATP hydrolysis?

It shields negative charges of phosphates and polarizes P–O bonds, making terminal phosphate more reactive.

What are the main factors affecting enzyme activity?

pH, temperature, enzyme concentration, substrate concentration, inhibitors, cofactors.

How does pH affect enzymes?

Changes protonation of catalytic groups and substrate; extreme pH can denature enzyme.

What is the optimum pH for pepsin?

About pH 2.

What is the optimum pH for trypsin?

About pH 8.

How does temperature affect enzymes?

Activity increases until optimum temperature, after which denaturation reduces activity.

What is the optimum temperature for human enzymes?

35–40°C.

What is the effect of enzyme concentration on Vmax?

Directly proportional; doubling enzyme doubles Vmax if substrate is abundant.

What is the effect of substrate concentration on velocity?

Hyperbolic increase until saturation; plateaus at Vmax.

What is Vmax?

The maximal velocity of an enzyme-catalyzed reaction at saturating substrate concentration.

What is Km?

The substrate concentration at which velocity is half of Vmax.

What does a low Km indicate?

High affinity of enzyme for substrate.

What does a high Km indicate?

Low affinity of enzyme for substrate.

What is kcat?

The turnover number: number of substrate molecules converted per enzyme molecule per unit time at saturation.

What is catalytic efficiency?

kcat/Km, a measure of enzyme performance.

What is the Michaelis–Menten equation?

v = (Vmax [S]) / (Km + [S]).

What is an International Unit (IU) of enzyme activity?

Amount of enzyme converting 1 µmol substrate per minute under defined conditions.

What is a katal?

Amount of enzyme converting 1 mol substrate per second.

What is specific activity?

Enzyme activity per mg protein, used to measure enzyme purity.

What are enzyme inhibitors?

Molecules that reduce enzyme activity by binding to enzyme.

What is competitive inhibition?

Inhibitor binds active site; Km increases, Vmax unchanged.

What is noncompetitive inhibition?

Inhibitor binds allosteric site; Km unchanged, Vmax decreases.

What is uncompetitive inhibition?

Inhibitor binds only ES complex; both Km and Vmax decrease.

What is irreversible inhibition?

Inhibitor covalently binds to enzyme, permanently inactivating it.

What are group-specific reagents?

Chemicals that react with specific amino acid side chains (e.g., iodoacetamide modifies cysteine).

What are affinity labels?

Substrate analogs that covalently modify active site residues.

What are suicide inhibitors?

Inhibitors activated by the enzyme itself to irreversibly inactivate it.

What are transition-state analogs?

Stable compounds resembling the transition state, binding enzyme tightly as potent inhibitors.

How does aspirin inhibit COX enzyme?

Acetylates Ser530 irreversibly, blocking prostaglandin synthesis.

What are the Lineweaver–Burk plot axes?

1/v versus 1/[S].

How does competitive inhibition appear on Lineweaver–Burk?

Lines intersect at y-axis; slope increases.

How does noncompetitive inhibition appear on Lineweaver–Burk?

Lines intersect at x-axis; Vmax decreases.

How does uncompetitive inhibition appear on Lineweaver–Burk?

Parallel lines; both Km and Vmax decrease.

What is a sequential multi-substrate reaction?

Both substrates must bind before any product is released.

What is a ping-pong reaction?

One product is released before the second substrate binds (enzyme temporarily modified).

What is proteolytic activation?

Activation of zymogens by cleavage of specific peptide bonds.

Give an example of proteolytic activation.

Trypsinogen → trypsin by enteropeptidase.

What is covalent modification?

Reversible attachment/removal of groups (e.g., phosphorylation) to regulate enzyme activity.

What is allosteric regulation?

Regulation by effector molecules binding sites other than active site, changing enzyme conformation.

What is a positive allosteric effector?

Molecule that increases enzyme activity upon binding.

What is a negative allosteric effector?

Molecule that decreases enzyme activity upon binding.

What are control proteins?

Regulatory proteins that bind enzymes to stimulate or inhibit their activity.

Give two examples of covalent modifications.

Phosphorylation and acetylation.

Give an example of allosteric enzyme kinetics.

Hemoglobin (sigmoidal O2 binding curve).