Enzyme Inhibition and Regulation: Kinetics, Allostery & ATCase

What is DFP?

Diisopropylfluorophosphate, an irreversible inhibitor that reacts with active-site serines.

How does DFP inhibit chymotrypsin?

It covalently modifies Ser195, preventing its normal catalytic action.

What is a suicide inhibitor?

A mechanism-based inhibitor that the enzyme converts into a reactive species that inactivates it.

Why can suicide inhibitors be selective?

Their activation depends on the targeted enzyme's own catalytic mechanism.

How can irreversible inhibitors help researchers?

They identify important catalytic residues or provide information about an enzyme's mechanism.

What is competitive inhibition?

Inhibitor binding to free enzyme excludes productive substrate binding.

Where do the classic competitive inhibitors bind?

At the active site, competing directly with substrate.

Which enzyme form binds a competitive inhibitor in the basic model?

Free enzyme, E.

What happens to apparent KM with competitive inhibition?

It increases.

What happens to Vmax with competitive inhibition?

It remains unchanged.

Why does competitive inhibition increase apparent KM?

More substrate is required to reach half of Vmax.

Why can high [S] overcome classic reversible competitive inhibition?

Substrate outcompetes inhibitor for enzyme binding.

What is the competitive inhibitor example in the notes?

Malonate competitively inhibits succinate dehydrogenase because it resembles succinate.

What is uncompetitive inhibition?

The inhibitor binds only the enzyme-substrate complex, ES.

Which complex forms during uncompetitive inhibition?

ESI: enzyme-substrate-inhibitor complex.

What happens to Vmax in uncompetitive inhibition?

It decreases.

What happens to apparent KM in uncompetitive inhibition?

It decreases by the same factor as Vmax.

Why does apparent KM decrease in uncompetitive inhibition?

Binding to ES shifts the kinetic balance toward bound complexes.

Can excess substrate fully overcome uncompetitive inhibition?

No.

What is mixed inhibition?

An inhibitor binds both free enzyme and ES with potentially different affinities.

Does a mixed inhibitor have to occupy the substrate-binding site?

No.

What happens to Vmax in mixed inhibition?

It decreases.

What can happen to apparent KM in mixed inhibition?

It increases, decreases, or stays unchanged, depending on relative affinity for E and ES.

When does mixed inhibition increase apparent KM?

When inhibitor binds free E more strongly than ES.

When does mixed inhibition decrease apparent KM?

When inhibitor binds ES more strongly than free E.

What is pure noncompetitive inhibition?

A special case of mixed inhibition in which the inhibitor binds E and ES equally strongly.

What happens to Vmax in pure noncompetitive inhibition?

It decreases.

What happens to KM in pure noncompetitive inhibition?

It remains unchanged.

Can high [S] restore the uninhibited Vmax in mixed or pure noncompetitive inhibition?

No.

What is Ki?

The dissociation constant for inhibitor binding to free enzyme.

What is Ki′?

The dissociation constant for inhibitor binding to ES.

What does a low Ki mean?

Tight inhibitor binding to free enzyme.

What does a low Ki′ mean?

Tight inhibitor binding to ES.

What is α?

α = 1 + [I]/Ki; it describes the effect of inhibitor binding to free enzyme.

What is α′?

α′ = 1 + [I]/Ki′; it describes the effect of inhibitor binding to ES.

What are the units of α and α′?

They are dimensionless.

What is the general mixed-inhibition rate equation?

v₀ = Vmax[S]/(αKM + α′[S]).

What is apparent Vmax in the mixed-inhibition model?

Vmax,app = Vmax/α′.

What is apparent KM in the mixed-inhibition model?

KM,app = (α/α′)KM.

What values describe competitive inhibition in the general model?

α > 1 and α′ = 1.

What values describe uncompetitive inhibition in the general model?

α = 1 and α′ > 1.

What values describe pure noncompetitive inhibition?

α = α′ > 1, equivalent to Ki = Ki′.

What is the competitive rate equation?

v₀ = Vmax[S]/(αKM + [S]).

What is the uncompetitive rate equation?

v₀ = Vmax[S]/(KM + α′[S]).

What is the general inhibited Lineweaver-Burk equation?

1/v₀ = (αKM/Vmax)(1/[S]) + α′/Vmax.

What is the inhibited Lineweaver-Burk slope?

αKM/Vmax.

What is the inhibited Lineweaver-Burk y-intercept?

α′/Vmax.

What is the inhibited Lineweaver-Burk x-intercept?

−α′/(αKM), which equals −1/KM,app.

What is the Lineweaver-Burk pattern for competitive inhibition?

Lines intersect on the y-axis; slope increases and x-intercept moves toward zero.

What is the Lineweaver-Burk pattern for uncompetitive inhibition?

Parallel lines; y-intercept rises and x-intercept becomes more negative.

Why are uncompetitive Lineweaver-Burk lines parallel?

KM and Vmax decrease by the same factor.

What is the Lineweaver-Burk pattern for pure noncompetitive inhibition?

Lines intersect on the x-axis; y-intercept and slope increase.

What is the Lineweaver-Burk pattern for mixed inhibition with unequal affinities?

Lines intersect left of the y-axis, usually above or below the x-axis.

How can kinetic data suggest competitive inhibition?

Vmax stays constant while apparent KM increases.

How can kinetic data suggest uncompetitive inhibition?

Vmax and apparent KM both decrease by the same factor.

How can kinetic data suggest pure noncompetitive inhibition?

Vmax decreases while KM stays constant.

How can kinetic data suggest unequal-affinity mixed inhibition?

Vmax decreases while apparent KM changes by a different factor.

What is an allosteric site?

A regulatory binding site distinct from the active site.

What is allosteric regulation?

Effector binding changes protein conformation and alters activity or ligand affinity.

What is a covalent modification?

Addition or removal of a chemical group through covalent bonds.

Name common regulatory covalent modifications.

Phosphorylation, adenylylation, uridylylation, methylation, and acetylation.

What is phosphorylation?

Addition of a phosphate group to a molecule.

What is adenylylation (AMPylation)?

Addition of an AMP group, usually using ATP as the donor.

What is uridylylation?

Addition of a UMP group, usually using UTP as the donor.

What is methylation?

Addition of a methyl group to a molecule.

What is adenylylation?

Addition of an AMP group, usually using ATP as the donor.

What is acetylation?

Addition of an acetyl group.

What does a protein kinase do?

Transfers a phosphate group, usually from ATP, to a protein.

What is the common protein kinase reaction?

Protein-OH + ATP → protein-O-phosphate + ADP.

Which ATP phosphate is transferred by a typical protein kinase?

The terminal γ phosphate.

Which amino acid side chains commonly undergo phosphorylation?

Serine, threonine, and tyrosine.

What does a protein phosphatase do?

Hydrolyzes a phosphate ester to remove phosphate from a protein.

What is the common protein phosphatase reaction?

Phosphoprotein + H₂O → dephosphorylated protein + inorganic phosphate.

Does a phosphatase transfer the removed phosphate back to ADP to make ATP?

No, it releases inorganic phosphate through hydrolysis.

Why can phosphorylation strongly affect protein structure?

It adds bulky negative charge and new hydrogen-bonding interactions.

How can phosphorylation link regulation to the cell's energy state?

ATP supplies the phosphate, connecting regulatory reactions with nucleotide availability.

Why can kinase pathways amplify signals?

One activated kinase can modify many target molecules, including other kinases.

Why is phosphorylation useful as a switch?

Kinases add phosphate and phosphatases remove it, allowing adjustable, reversible regulation.

Does phosphorylation always mean the protein is active?

No, its effect is specific to the protein and phosphorylation site.

What is proteolysis?

Cleavage of peptide bonds in a protein.

What is a proenzyme?

An inactive enzyme precursor that can be converted to an active enzyme.

What is a zymogen?

An inactive enzyme precursor, especially an inactive protease precursor.

How does proteolytic cleavage activate a zymogen?

It produces a structural rearrangement that makes the active site functional.

Why are digestive proteases synthesized as zymogens?

To limit unwanted digestion of proteins before the enzymes reach the proper location.

What is chymotrypsinogen?

The inactive precursor of chymotrypsin.

How is chymotrypsinogen initially activated?

Trypsin cleaves a specific peptide bond; further processing produces mature chymotrypsin.

How does proteolytic activation differ from phosphorylation?

Cleavage is generally irreversible; phosphorylation can be reversed by phosphatases.

Can proteolysis also inactivate a protein?

Yes, cleavage or degradation can destroy function.

What is feedback inhibition?

A pathway's end product inhibits an earlier enzyme, limiting further product synthesis.

Why is feedback inhibition useful?

It prevents unnecessary product accumulation and conserves resources.

What is an allosteric enzyme?

An enzyme whose activity is altered by binding of regulatory effectors at allosteric sites.

Are all allosteric enzymes multimeric and sigmoidal?

No, those are common features but not universal requirements.

What does a sigmoidal velocity-versus-substrate curve suggest?

Positive cooperativity among substrate-binding sites.

How does a cooperative allosteric kinetic curve differ from a Michaelis-Menten curve?

It is often sigmoidal rather than hyperbolic.

Why use K0.5 or S0.5 for many allosteric enzymes?

It names the substrate concentration giving half-maximal rate without assuming Michaelis-Menten kinetics.

What is the T state of the allosteric enzymes discussed here?

A less active, lower-affinity conformation.

What is the R state of the allosteric enzymes discussed here?

A more active, higher-affinity conformation.

How can an allosteric activator affect a kinetic curve?

It can favor R, shift the curve left, and/or increase maximal activity.

How can an allosteric inhibitor affect a kinetic curve?

It can favor T, shift the curve right, and/or decrease maximal activity.

What does ATCase stand for?

Aspartate transcarbamoylase.