Exam 2 BIOC 3022 Lecture 14

How can we describe how enzymes behave?

Let us predict V0 at a given [S] and compare enzymes, kcat, Vmax, Km, and kcat/Km describe how an enzyme behaves

What are inhibitors?

Inhibitors are compounds that decrease enzyme's activity

Irreversible inhibitors

Inactivators that react with the enzyme, one inhibitor molecule can permanently shut off one enzyme molecule. Often powerful toxins.

Reversible inhibitors

Can bind to and can dissociate from the enzyme, can be structural analogs of substrates or products. Often used as drugs to slow down a specific enzyme.

Where can a reversible inhibitor bind?

To the free enzyme and prevent the binding of the substrate/reaction. To the enzyme-substrate complex and prevent the reaction.

Penicillin

An irreversible inhibitor. It resembles the substrate and binds the active site. Forms a covalent bond with the enzyme. The enzyme cannot build the bacterial cell wall. Suicide inhibitor: irreversible

Competitive reversible inhibitors

Directly competes with substrate fro active site. When the inhibitor is bound to the active site, substrate cannot bind.

Uncompetitive reversible inhibitors

Binds (only) the ES complex at an allosteric site and prevents catalysis. Does not affect substrate binding.

Mixed reversible inhibitors

Binds enzyme with or without substrate and negatively affects both substrate binding and catalysis

Noncompetitive reversible inhibitors

Binds enzyme with or without substrate and blocks catalysis and leaves substrate binding intact (special case of mixed)

What does the competitive inhibitor do?

Does not affect catalysis directly and does not react to form product.

Competitive inhibition's Vmax and Km

Vmax is unchanged and apparent Km increases

What does the competitive inhibition's lineweaver-burk plot look like?

Lines meet on the y-axis

What does the uncompetitive inhibition do?

Does not stop substrate binding and blocks catalysis.

Uncompetitive inhibition's Vmax and Km

Vmax and Km both decrease

Km/Vmax is unchanged

What does the uncompetitive inhibitor's lineweaver-burk plot look like?

Parallel lines

What does the mixed inhibition do?

Binds E or ES (affinity differs) at a site other than the active site. Inhibits both substrate binding and catalysis.

Mixed inhibition's Vmax and Km

Vmax decreases and apparent Km can increase or decrease.

What does the mixed inhibition's lineweaver-burk plot look like?

Lines meet left of the y-axis

What does the noncompetitive inhibition do?

Binds E and ES with same affinity. Binds a site other than the active site. Blocks catalysis and leaves substrate binding intact.

Noncompetitive inhibition's Vmax and Km

Vmax decreases and Km is unchanged

What does the noncompetitive inhibition's lineweaver-burk plot look like?

Lines meet on the x-axis

Methotrexate

Resembles the folate substrate of DHFR. Binds the active site where folate would bind.

Methotrexate's Vmax and Km

More substrate competes it away: Vmax is unchanged, apparent Km increases

Which inhibition can be overcome by adding more substrate, and why?

Competitive inhibition can be overcome by adding more substrate because the substrate can outcompete and displace the inhibitor from the enzyme's active site.

Allosteric enzymes

Binding at one site changes activity at another site. V0 vs [S] is sigmoidal instead of hyperbolic.

Allosteric enzymes T-state

Low affinity for substrate

Allosteric enzymes R-state

High affinity

Allosteric enzymes homotropic effector

the substrate

Allosteric enzymes heterotropic effector

not the substrate

n = 1

Hyperbolic curve, no cooperatively

n > 1

positive cooperativity, sigmoidal curve

n < 1

negative cooperativity

What homotropic substrate favors the R-state?

Aspartate

What activator favors the R-state?

ATP

What inhibitor favors the T-state?

CTP

Heterotropic effectors

ATP and CTP are not substrates