bios 352 ch.6

What is an enzyme?

A biological catalyst that accelerates a reaction without being consumed.

What are most enzymes made of?

Proteins.

What is a ribozyme?

An RNA molecule with catalytic activity.

What is a substrate?

The reactant on which an enzyme acts.

What is an active site?

The region of an enzyme where substrate binding and catalysis occur.

What is enzyme specificity?

The ability of an enzyme to recognize particular substrates and reactions.

What is activation energy (ΔG‡)?

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

How do enzymes accelerate reactions?

They lower activation energy by providing a more favorable reaction pathway.

Do enzymes change the equilibrium constant?

No; they accelerate forward and reverse reactions without changing equilibrium.

Do enzymes change the overall reaction ΔG?

No; the free-energy difference between reactants and products remains unchanged.

What is the transition state?

A high-energy, unstable arrangement of atoms along the reaction pathway.

What is transition-state stabilization?

Preferential binding interactions that lower the energy of the transition state.

What did Linus Pauling propose about enzyme catalysis?

Enzymes bind transition states more strongly than substrate ground states.

What is binding energy (ΔGB)?

Free energy released through favorable enzyme-substrate interactions.

How does binding energy contribute to catalysis?

It helps offset the energy needed to reach the transition state.

How does an enzyme reduce substrate entropy?

By restricting substrate movement and orienting reacting groups.

What is substrate desolvation?

Removal or rearrangement of surrounding water when substrate binds the active site.

What is induced fit?

A conformational change in the enzyme upon substrate binding.

How does induced fit promote catalysis?

It positions catalytic groups and helps stabilize the transition state.

What is a cofactor?

A nonprotein component required for the activity of some enzymes.

What are two major types of cofactors?

Metal ions and organic coenzymes.

What is a coenzyme?

An organic cofactor that assists enzyme catalysis.

What is a prosthetic group?

A cofactor tightly or permanently associated with an enzyme.

What is an apoenzyme?

The enzyme protein without its required cofactor.

What is a holoenzyme?

The complete, catalytically active enzyme with its required cofactors.

What is general acid catalysis?

A catalytic group donates a proton to facilitate a reaction.

What is general base catalysis?

A catalytic group accepts a proton to facilitate a reaction.

What is general acid-base catalysis?

Enzyme-assisted proton transfer during the reaction.

Which amino acid often participates in acid-base catalysis?

Histidine.

Why is histidine useful in acid-base catalysis?

Its side chain can donate or accept protons near physiological pH.

What is covalent catalysis?

Formation of a temporary covalent bond between enzyme and substrate.

What does covalent catalysis change?

The reaction pathway through a transient covalent intermediate.

What does covalent catalysis require?

A reactive nucleophilic group on the enzyme.

Which enzyme groups can participate in covalent catalysis?

Serine hydroxyl, cysteine thiolate, amino, or carboxylate groups.

What is metal ion catalysis?

Catalysis assisted by a bound metal ion.

How can metal ions assist catalysis?

By stabilizing charge, activating water, or participating in redox reactions.

What is electrostatic catalysis?

Stabilization of transition-state charge by favorable electrostatic interactions.

What is the difference between metal ion and electrostatic catalysis?

Metal ion catalysis specifically involves metal ions; electrostatic catalysis involves charge interactions more generally.

What happens to an enzyme after a complete catalytic cycle?

It returns to its original functional state.

What is chymotrypsin?

A serine protease that hydrolyzes peptide bonds.

What types of amino acid side chains does chymotrypsin preferentially recognize?

Bulky aromatic side chains.

What is the catalytic triad of chymotrypsin?

Ser195, His57, and Asp102.

What is the role of Ser195?

It acts as a nucleophile and forms a transient acyl-enzyme intermediate.

What is the role of His57?

It acts as a general acid and general base during catalysis.

What is the role of Asp102?

It helps orient and stabilize His57.

What is the oxyanion hole?

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

How does the oxyanion hole stabilize intermediates?

Through hydrogen bonds to the negatively charged oxygen.

What are the two phases of chymotrypsin catalysis?

Acylation and deacylation.

What occurs during chymotrypsin acylation?

Ser195 attacks the peptide carbonyl and an acyl-enzyme intermediate forms.

What occurs during chymotrypsin deacylation?

Activated water attacks the acyl-enzyme, releasing product and regenerating Ser195.

What happens in the first step of chymotrypsin catalysis?

The peptide substrate binds in the active site.

What does His57 do during the initial Ser195 attack?

It accepts a proton from Ser195, activating the serine oxygen.

What attacks the peptide carbonyl during acylation?

The oxygen of Ser195.

What is the first tetrahedral intermediate?

The intermediate formed after Ser195 attacks the peptide carbonyl.

What happens when the first tetrahedral intermediate collapses?

The peptide bond breaks, the amine product leaves, and the acyl-enzyme forms.

What attacks the acyl-enzyme during deacylation?

A water molecule activated by His57.

What is the second tetrahedral intermediate?

The intermediate formed when activated water attacks the acyl-enzyme carbonyl.

What happens when the second tetrahedral intermediate collapses?

The carboxyl-containing product leaves and Ser195 is regenerated.

Which catalytic mechanisms does chymotrypsin use?

General acid-base, covalent, and electrostatic catalysis.

What is enzyme kinetics?

The study of enzyme-catalyzed reaction rates.

What is initial velocity (V0)?

The reaction rate measured near the beginning of a reaction.

Why measure initial velocity?

Substrate depletion and product accumulation are minimal.

What is the enzyme-substrate complex (ES)?

The intermediate formed when substrate binds enzyme.

What is the simple Michaelis-Menten reaction scheme?

E + S ⇌ ES → E + P.

What is the steady-state assumption?

ES concentration remains approximately constant during initial-rate measurements.

What is the Michaelis-Menten equation?

V0 = Vmax[S]/(Km + [S]).

What is Vmax?

The maximum initial velocity approached at saturating substrate concentration.

What is Km?

The substrate concentration at which V0 equals half of Vmax.

What is V0 when [S] = Km?

0.5Vmax.

What happens when [S] is much greater than Km?

V0 approaches Vmax because the enzyme is nearly saturated.

What happens when [S] is much smaller than Km?

V0 ≈ Vmax[S]/Km; velocity depends approximately linearly on [S].

When should the full Michaelis-Menten equation be used?

When [S] is not clearly much larger or much smaller than Km.

What happens to Vmax when active enzyme concentration doubles?

Vmax doubles if kcat stays constant.

What happens to Km when enzyme concentration doubles?

Km remains unchanged under otherwise identical conditions.

What is the relationship between Km and microscopic rate constants?

Km = (k-1 + kcat)/k1 for a simple one-substrate mechanism.

When does Km approximate the ES dissociation constant Kd?

When kcat is much smaller than k-1.

Does lower Km always prove stronger substrate binding?

No; Km approximates binding affinity only under appropriate kinetic assumptions.

What is kcat?

The turnover number: substrate molecules converted per active site per unit time at saturation.

What is the equation for kcat?

kcat = Vmax/[Et].

What is catalytic efficiency?

The specificity constant kcat/Km.

What does a larger kcat/Km indicate?

Greater catalytic efficiency, particularly at low substrate concentration.

What is a Lineweaver-Burk plot?

A double-reciprocal plot of 1/V0 versus 1/[S].

What is the Lineweaver-Burk equation?

1/V0 = (Km/Vmax)(1/[S]) + 1/Vmax.

What is the y-intercept of a Lineweaver-Burk plot?

1/Vmax.

What is the x-intercept of a Lineweaver-Burk plot?

-1/Km.

What is the slope of a Lineweaver-Burk plot?

Km/Vmax.

How can Km be calculated from the Lineweaver-Burk slope and y-intercept?

Km = slope/y-intercept.

What is a reversible inhibitor?

A compound that decreases enzyme activity through reversible binding.

What is competitive inhibition?

An inhibitor competes with substrate for binding to free enzyme.

How does competitive inhibition affect apparent Km?

Apparent Km increases.

How does competitive inhibition affect Vmax?

Vmax remains unchanged.

Can competitive inhibition be overcome by high [S]?

Yes, for a reversible competitive inhibitor.

Where do competitive inhibition lines intersect on a Lineweaver-Burk plot?

On the y-axis.

What is uncompetitive inhibition?

An inhibitor binds the enzyme-substrate complex rather than free enzyme.

How does uncompetitive inhibition affect Km and Vmax?

Both apparent Km and Vmax decrease proportionally.

What do uncompetitive inhibition lines look like on a Lineweaver-Burk plot?

Parallel lines.

What is mixed inhibition?

An inhibitor binds free enzyme and ES with different affinities.

How does mixed inhibition affect Vmax?

Apparent Vmax decreases.

How can mixed inhibition affect apparent Km?

It can increase or decrease depending on inhibitor binding preference.

What is pure noncompetitive inhibition?

A special case of mixed inhibition with equal inhibitor affinity for E and ES.