Enzyme Inhibition

Reversible Inhibitors

interact with the enzyme through noncovalent interactions

associate and dissociate with the enzyme

Irreversible inhibitors

form stable covalent bonds with enzymes

block the active site from binding substrate

consequently, causes a decrease in the concentration of enzyme active sites

4 categories of reversible inhibitors

competitive, uncompetitive, noncompetitive, mixed

competitive inhibitors

compete with substrates for the same binding site on the enzyme - inhibitor is prevented from binding to the active site if the active site is already occupied by a substrate molecule

• increasing the substrate concentration favors the binding of substrate to the enzyme over the inhibitor (I)

competitive inhibitor Ki equation

Ki = [I] / ((mi/mo) -1)

dissociation constant will have the same units of concentration as the inhibitor’s concentration

which line do you evaluate for Km and Vmax vs Km apparent and Vmax apparent

for Km and Vmax evaluate the top line

for Km apparent and Vmax apparent evaluate the bottom line

for competitive inhibition of Km and Vmax - what is affected

Vmax is unaffected

as more and more inhibitor is added the Km apparent increases

noncompetitive inhibition

when the binding of the inhibitor has no effect on the binding of substrate

I and S bind to different sites on the enzyme such that the binding of I has no effect on the binding of S

decrease the maximum rate of reaction without affecting Km - inhibitor inhibits a fraction the enzyme regardless of the substrate concentration

slope formula for line weaver burke plots - all except uncompetitive inhibition

Km/Vmax (1+ [I]/Ki)

y-intercept formula for line weaver burke plots

1/Vmax (1+ [I]/Ki)

mixed noncompetitive inhibition

it is rare due to allosteric nature of ligand binding, that the binding of substrate does not affect the binding of inhibitor (mixed noncompetitive more likely than pure noncompetitive)

→ even when the inhibitor binds to a separate site on the enzyme, the binding of substrate is communicated to that site by conformational changes

→ when the substrate binds the active site, the enzyme shifts shape a little and that shift reaches the inhibitor site

→ the inhibitor’s site looks slightly different on ES than on free E and the inhibitor binds ES more or less tightly than E and vice-versa for inhibitor binding and shape change of active sites

for what type of inhibition does Ki not equal Ki’

mixed noncompetitive inhibition

for mixed noncompetitive inhibition are Vmax or Km affected

yes both are affected - effect varies based on Ki < Ki’ or Ki > Ki’

how do you calculate the Ki for mixed noncompetitive inhibitors

Ki = [I] / ((mi/mo) -1)

how do you calculate the Ki’ for mixed noncompetitive inhibition

Ki’ = [I]/((bi/bo) -1)

uncompetitive inhibition

inhibitor only binds to the enzyme - substrate complex and not to the free enzyme

→ common for enzymes where during enzyme catalyzed rxns - the enzyme or coenzyme becomes covalently attached to the substrate

→ both the Km apparent and Vmax apparent are decreased by the same amount in the presence of the inhibitor

for uncompetitive inhibition what do the line weaver burke plots look like

series of parallel lines

slope formula for line weaver burke plots of uncompetitive inhibition

Km/Vmax

how to calculate Ki for uncompetitive inhibition

Ki = [I] / ((bi/bo) - 1)

irreversible inhibition

covalently modify the enzymes which destroys catalytic activity

the Lineweaver - burke plot for irreversible inhibitors cannot be distinguished from the noncovalent inhibition pattern, because of the net loss of active enzyme

irreversible inhibition can be distinguished from noncompetitive inhibition by measuring the time dependent loss of enzyme activity

dialysis does not restore activity as it would for a noncovalent inhibitor