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Biochemistry Unit 5: Enzyme Inhibition and Regulation

Cells control enzymes, and drugs block them. This unit covers the kinetic signatures of reversible inhibitors, irreversible inhibitors, and the main ways cells regulate enzymes: allostery, covalent modification, cleavage and feedback.

Covers: Competitive inhibition, Uncompetitive inhibition, Noncompetitive and mixed inhibition, Irreversible and suicide inhibitors, Allosteric regulation, Covalent modification, Zymogens and feedback control

Study guide

Reversible inhibitors bind noncovalently and can leave. A competitive inhibitor binds the free enzyme's active site, often because it looks like the substrate. It raises the apparent Km by a factor α = 1 + [I]/Ki but leaves Vmax unchanged, because enough substrate can outcompete it. On a Lineweaver-Burk plot the lines cross on the y-axis. Methotrexate, a folate analog, and ethanol used against methanol poisoning are classic examples. To find Ki from data, compute α = Km,app/Km and solve for Ki.

An uncompetitive inhibitor binds only the ES complex. It lowers both Vmax and Km by the same factor α′ = 1 + [I]/Ki′, so Lineweaver-Burk lines are parallel. A mixed inhibitor binds both E and ES, but with different affinities, so both Km and Vmax change; if it prefers free E, Km rises, and if it prefers ES, Km falls. The special case of equal binding is pure noncompetitive inhibition: Vmax falls by α and Km stays the same, and lines cross on the x-axis. Substrate cannot overcome uncompetitive, mixed or noncompetitive inhibition.

Irreversible inhibitors form covalent bonds or bind so tightly they never leave. Aspirin acetylates a serine in cyclooxygenase, and nerve agents like sarin modify the active-site serine of acetylcholinesterase. Suicide (mechanism-based) inhibitors are unreactive until the enzyme begins its normal reaction on them, then trap the enzyme; penicillin inactivates bacterial transpeptidase this way. Because the enzyme is removed from action, adding substrate does not help, and recovery requires new enzyme synthesis. Kinetically, an irreversible inhibitor simply lowers the amount of working enzyme, so Vmax falls while the Km of the remaining active molecules is unchanged.

Cells regulate enzymes in several ways. Allosteric enzymes have regulatory sites separate from the active site and usually several subunits, giving sigmoidal kinetics. Activators stabilize the high-activity R state and inhibitors stabilize the T state. Feedback inhibition, in which an end product inhibits the committed step, is common: CTP inhibits aspartate transcarbamoylase, while ATP activates it. PFK-1 in glycolysis is inhibited by ATP and citrate and activated by AMP and fructose 2,6-bisphosphate, so it tracks the energy state. On graphs, remember that allosteric enzymes do not follow Michaelis-Menten kinetics, so the term K0.5 is used for the substrate level giving half-maximal rate.

Covalent modification, most often phosphorylation by kinases and removal by phosphatases, switches enzymes on or off in seconds. Hormones such as glucagon and epinephrine work through cAMP and protein kinase A, phosphorylating glycogen phosphorylase (activating it) and glycogen synthase (inactivating it). Proteolytic activation is irreversible: zymogens such as trypsinogen, chymotrypsinogen and pepsinogen, and blood-clotting factors, are made inactive and cleaved when and where they are needed. Cleavage cascades amplify signals, so a small trigger activates many enzyme molecules.

Key ideas

• Competitive inhibitors raise Km and leave Vmax unchanged.
• Uncompetitive inhibitors lower Km and Vmax equally and give parallel double-reciprocal lines.
• Mixed inhibitors change both Km and Vmax; pure noncompetitive ones lower only Vmax.
• Irreversible and suicide inhibitors permanently inactivate enzyme molecules.
• Allosteric effectors shift enzymes between T and R states, moving sigmoid curves.
• Phosphorylation is a fast, reversible switch controlled by kinases and phosphatases.
• Zymogens are activated irreversibly by cleavage, often in amplifying cascades.

Formulas and rules

Common mistakes

• Saying competitive inhibitors lower Vmax; they leave it unchanged.
• Forgetting that uncompetitive inhibitors lower Km (apparent affinity rises).
• Treating α as [I]/Ki instead of 1 + [I]/Ki.
• Assuming excess substrate can reverse an irreversible inhibitor.
• Mixing up Lineweaver-Burk patterns: competitive crosses on the y-axis, noncompetitive on the x-axis, uncompetitive is parallel.

Before the test, make sure you can

• Identify inhibitor type from changes in Km and Vmax.
• Identify inhibitor type from Lineweaver-Burk patterns.
• Calculate α, apparent Km, apparent Vmax and Ki.
• Calculate rates in the presence of inhibitors.
• Explain irreversible and suicide inhibition with drug examples.
• Explain allosteric regulation and feedback inhibition with examples.
• Explain how phosphorylation and zymogen activation control enzymes.
• Apply IC50 and Ki relationships for competitive inhibitors.

Words to know

Competitive inhibitor

Binds the free enzyme's active site; raises Km, Vmax unchanged.

Uncompetitive inhibitor

Binds only ES; lowers Km and Vmax by the same factor.

Mixed inhibitor

Binds E and ES with different affinities; changes both Km and Vmax.

Pure noncompetitive inhibitor

Binds E and ES equally; lowers Vmax, Km unchanged.

Ki

The dissociation constant of the enzyme-inhibitor complex; smaller means tighter binding.

IC50

The inhibitor concentration that reduces activity by half under given assay conditions.

Irreversible inhibitor

Covalently or permanently inactivates an enzyme, such as aspirin or sarin.

Suicide inhibitor

A mechanism-based inhibitor activated by the enzyme's own catalysis, such as penicillin.

Allosteric site

A regulatory site distinct from the active site where effectors bind.

Allosteric activator

An effector that stabilizes the R state and shifts the curve left.

Feedback inhibition

Inhibition of an early pathway enzyme by the pathway's end product.

Committed step

The first irreversible step unique to a pathway, usually the regulated one.

Protein kinase

An enzyme that transfers phosphate from ATP to a protein's Ser, Thr or Tyr.

Phosphatase

An enzyme that removes phosphate groups from proteins.

Zymogen

An inactive enzyme precursor activated by proteolytic cleavage.

Enteropeptidase

An intestinal protease that activates trypsinogen to trypsin.

Aspartate transcarbamoylase

An allosteric enzyme of pyrimidine synthesis inhibited by CTP and activated by ATP.

Sample practice questions

30 more questions like these, from level 1 to 5, are in the practice set.