Enzyme fundamentals, mechanisms, and regulation

What is an enzyme?

A biological catalyst that increases reaction rate without being consumed.

What are the three defining features of enzymes?

Catalytic power, specificity, and regulation.

What is catalytic power?

The ability of an enzyme to accelerate a reaction compared with the uncatalyzed reaction.

What is enzyme specificity?

The ability to recognize particular substrates and catalyze specific reactions.

What is the active site?

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

What is the enzyme–substrate (ES) complex?

The temporary complex formed when substrate binds to an enzyme.

What is the induced-fit model?

Substrate binding causes a conformational change in the enzyme that promotes catalysis.

What are the six traditional enzyme classes?

Oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases.

oxidoreductases

Catalyze oxidation–reduction reactions involving electron transfer.

transferases

Transfer functional groups between molecules.

hydrolases

Break chemical bonds using water.

lyases

Break or form bonds without hydrolysis or oxidation, often creating or removing double bonds.

isomerases

Rearrange atoms within a molecule to form an isomer.

ligases

Join two molecules together, usually using energy from ATP hydrolysis.

What is a cofactor?

A nonprotein component required for enzyme activity; it may be inorganic or organic.

What is a coenzyme?

An organic cofactor, often vitamin-derived, that participates in catalysis.

What is the difference between a cofactor and a coenzyme?

Cofactors can be inorganic or organic; coenzymes are specifically organic cofactors.

What is activation energy (ΔG‡)?

The free-energy barrier that reactants must overcome to reach the transition state.

What do enzymes change on a reaction coordinate diagram?

They lower activation energy (ΔG‡), increasing reaction rate.

What do enzymes NOT change?

Overall ΔG, the equilibrium constant, or the equilibrium position.

Do enzymes change whether a reaction is spontaneous?

No. They change reaction rate, not thermodynamic favorability.

What is the transition state?

A high-energy, unstable configuration formed as reactants convert into products.

How do enzymes stabilize the transition state?

By making favorable interactions with the transition state, lowering ΔG‡.

Why must an enzyme bind the transition state more favorably than the substrate?

Preferential transition-state binding lowers the activation barrier and accelerates catalysis.

What happens if an enzyme binds its substrate too tightly?

The ES complex becomes overly stabilized, making the transition state harder to reach and potentially slowing catalysis.

What is binding energy?

Free energy released through favorable interactions between an enzyme and a bound ligand.

How does binding energy contribute to catalysis?

It helps orient substrates, promote conformational changes, and stabilize the transition state.

What is ES destabilization?

Raising the energy of the enzyme–substrate complex relative to the transition state, making progression easier.

What is general acid–base catalysis?

Catalysis in which groups donate or accept protons during a reaction.

What is a general acid?

A group that donates H⁺ during catalysis.

What is a general base?

A group that accepts H⁺ during catalysis.

What is covalent catalysis?

Catalysis involving temporary covalent bond formation between enzyme and substrate.

What is metal-ion catalysis?

Metal ions assist reactions by stabilizing charges, orienting substrates, activating water, or participating in redox chemistry.

What is a low-barrier hydrogen bond?

An unusually strong hydrogen bond that may contribute to catalytic stabilization when donor and acceptor proton affinities are closely matched.

What type of enzyme is chymotrypsin?

A serine protease that hydrolyzes peptide bonds.

What amino acids does chymotrypsin preferentially cleave after?

Bulky aromatic residues: phenylalanine, tyrosine, and tryptophan.

What is the catalytic triad of chymotrypsin?

Ser195, His57, and Asp102.

What is the role of Ser195, His57, and Asp102.

Acts as the nucleophile and forms a temporary covalent bond with the substrate.

Acts as a general acid and base by accepting and donating protons.

Positions and stabilizes His57 during catalysis.

What are the two phases of the chymotrypsin mechanism?

Acylation and deacylation.

What happens first during chymotrypsin acylation?

His57 removes a proton from Ser195, activating its oxygen for nucleophilic attack.

What does Ser195 attack?

The carbonyl carbon of the peptide bond being cleaved.

What forms after Ser195 attacks?

The first tetrahedral intermediate.

What is the oxyanion hole?

An active-site region that stabilizes negatively charged oxygen in tetrahedral intermediates.

What happens when the first tetrahedral intermediate collapses?

The peptide bond breaks, the first product leaves, and an acyl-enzyme intermediate remains.

What is the acyl-enzyme intermediate?

A temporary covalent bond between the substrate's acyl group and Ser195.

What happens during deacylation?

His57 activates water, which attacks the acyl-enzyme carbonyl to form a second tetrahedral intermediate.

How is chymotrypsin regenerated?

The second tetrahedral intermediate collapses, releasing the remaining product and restoring Ser195.

How many tetrahedral intermediates form during chymotrypsin catalysis?

Two: one during acylation and one during deacylation.

Which catalytic strategies does chymotrypsin use?

Covalent catalysis and general acid–base catalysis, aided by transition-state stabilization.

How does an aspartyl protease differ from chymotrypsin?

An aspartyl protease activates water for direct attack rather than forming a covalent serine acyl-enzyme intermediate.

walk through the chymotrypsin mechanism step by step. in a few sinmple sentences

• Binding: The target protein binds to the enzyme, sliding its bulky amino acid side chain into a specific hydrophobic pocket.

• Nucleophilic Attack: A nearby histidine strips a proton from serine, turning that serine into a powerful nucleophile that attacks the protein's carbonyl carbon.

• First Cleavage (Acylation): An unstable intermediate forms and collapses, breaking the peptide bond; the first half of the broken protein leaves, while the other half remains covalently glued to the serine.

• Water Attack (Deacylation): A water molecule enters, and the histidine strips a proton from it to turn it into a hydroxide ion, which attacks the remaining protein-enzyme bond.

• Release and Reset: The second intermediate collapses, releasing the remaining protein fragment and resetting the enzyme back to its starting state.

What is allosteric regulation?

Binding of a regulatory molecule away from the active site changes enzyme activity.

What is an allosteric site?

A regulatory binding site separate from the active site.

What is positive cooperativity?

Binding of one ligand increases the affinity of other binding sites.

S shaped

What is negative cooperativity?

Binding of one ligand decreases the affinity of other binding sites.

hyperbolic curve

What is the MWC model?

A concerted model in which subunits switch together between pre-existing T and R states

ligand doesnt cause the change, it picks a winner

positive cooperativity

all subunits must be in the same state

T state?

Tense, typically lower-affinity or less active conformation.

R state

Relaxed, typically higher-affinity or more active conformation.

KNF model?

A sequential model in which ligand binding induces conformational changes in its neighboring individual subunits.

induced fit

alters the neighbors affinity for the change, can decrease or increase affinity —> positive or negative cooperativity allowed

mixed conformations allowed

What is the main difference between MWC and KNF?

MWC uses concerted conformational selection; KNF uses sequential induced conformational changes.

How does an allosteric activator typically shift a substrate-response curve?

Left, because less substrate is needed for a given response.

How does an allosteric inhibitor typically shift a substrate-response curve?

Right, because more substrate is needed for a given response.

Why can an activator make a sigmoidal curve more hyperbolic?

It favors the active R state before substantial substrate binding occurs.

What is feedback inhibition? Why is it useful?

A pathway's end product inhibits an earlier enzyme to prevent overproduction.

It conserves energy and prevents unnecessary synthesis of products.

What is a zymogen?

An inactive enzyme precursor activated through proteolytic cleavage.

Why are digestive enzymes produced as zymogens?

To prevent them from damaging the tissues that produce them.

Is proteolytic zymogen activation reversible?

no

What is covalent enzyme regulation?

Regulation by adding or removing covalently attached chemical groups.

What do protein kinases do?

Transfer phosphate groups from ATP to target proteins.

What do protein phosphatases do?

Remove phosphate groups from proteins.

Does phosphorylation always activate enzymes?

No. It can activate or inhibit depending on the enzyme.

covalent modification with cAMP and protein kinase A

Adenylyl cyclase converts ATP into cAMP.

•Reaction is driven forward by subsequent hydrolysis of pyrophosphate

•activated by G-protein subunit signal binding

•cAMP is therefore a secondary messenger (which binds to and activates protein kinase A)

What does glycogen phosphorylase do?

To break down glycogen when fuel is needed and conserve glycogen when energy is abundant.

What are the TWO major ways glycogen phosphorylase is regulated?

Allosteric regulation and covalent modification (phosphorylation).

How does allosteric regulation control glycogen phosphorylase?

Molecules bind to regulatory sites and change its activity without adding or removing phosphate groups.

Why does AMP activate muscle glycogen phosphorylase?

High AMP signals low cellular energy, so the muscle needs to break down glycogen for fuel.

Why does ATP inhibit muscle glycogen phosphorylase?

High ATP signals that the muscle already has sufficient energy.

Why does glucose-6-phosphate inhibit muscle glycogen phosphorylase?

It signals that glucose-derived fuel is already available.

What is the difference between phosphorylase a and phosphorylase b?

Phosphorylase a is phosphorylated and generally more active; b is dephosphorylated and less active.

kinase

Adds phosphate groups to glycogen phosphorylase, converting b into a.

What does PKA do in glycogen breakdown?

Phosphorylates and activates phosphorylase kinase, which then activates glycogen phosphorylase.

Why is glycogen phosphorylase an example of covalent modification?

Its activity changes when phosphate groups are covalently added or removed.

What is an isoenzyme?

A different molecular form of an enzyme that catalyzes the same reaction.

What is compartmentation?

Separation of enzymes and pathways into different cellular locations.

It organizes metabolism, enables localized regulation, and helps separate opposing pathways.