Enzyme Inhibition

four categories of reversible enzyme inhibitors

• competitive

• uncompetitive

• noncompetitive

• mixed inhibitors

competitive inhibitors

compete w/ the substrate for the same binding site on the enzyme

• the inhibitor is prevented from binding to the active site if the active site is already occupied by a substrate moleculed

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

what plot is used for evaluating competitive inhibition?

Lineweaver-Burk Plot

why do plots at various inhibitor concentrations pass through the same intercept corresponding to 1/Vmax?

b/c the effect of the inhibitor disappears as the substrate concentration becomes saturating

how is Ki determined using the Lineweaver-Burk Plot?

by measuring the slope of the plots as a function of the inhibitor concentration

specificity pocket of trypsin

can accommodate an arginine side chain of a polypeptide substrate or a benzamidine ion which compete w/ the substrate for the binding site and acts as a competitive inhibitor

Ki for a competitive inhibitor

Ki = [l] / (mi/m0 - 1)

noncompetitive inhibitors

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

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

Ki for a noncompetitve inhibitor equation

Km = [l] / (mi/m0 - 1) = [l] / bi/b0 - 1)

competitive inhibition: equation for v

v = Vmax[S]/({Km(1 + [I]/Ki) + [S])

competitive inhibition: why do the plots all pass through the same y-intercept corresponding to 1/Vmax?

b/c the effect of the inhibitor disappears as the substrate concentration becomes saturating

competitive inhibition: what do the slopes depend on?

the concentration of the inhibitor (Km/Vmax)(1+ [I]/Ki) compared to the slope Km/Vmax for the uninhibited reaction

competitive inhibition: how can we determine Ki

by measuring the slope of the plots as a function of the inhibitor concentration

noncompetitive inhibition: main characteristic

it decreases the maximum rate of the reaction without affecting the Km

• the inhibitor inhibits a function of the enzyme regardless of the substrate concentration

• Lineweaver-Burk plots all intersect the x-axis at the same point which corresponds to 1/Km

noncompetitive inhibition: y-intercept

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

mixed noncompetitive inhibition

when the binding of the inhibitor has effects on the binding of substrate

• inhibitor and substrate bind to different site on the enzyme such that the binding of the inhibitor effects the binding of the substrate

• Ki =/ Ki`

mixed noncompetitive inhibition equations

v = ( (V_max [S]) / (1 + [I]/K_i) ) / ( ( (1 + [I]/K_i) K_m ) / (1 + [I]/K_i) + [S] )

mixed noncompetitive inhibition: Ki and Ki` equations

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

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

uncompetitive inhibition

when the inhibitor only bind to the enzyme-substrate complex and not the free enzyme

• common for enzymes where during the catalyzed reaction the enzyme or a coenzyme becomes covalently attached to the substrate

• both Km and Vmax decrease by the same amount in the presence of the inhibitor

uncompetitive inhibition equations

y-int

uncompetitive inhibition: Ki equation

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

irreversible inhibition

covalently modify the enzyme which destroys catalytic activity

• cannot be distinguished by lineweaver-burk plot b/c of the net loss of active enzyme

• 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 inhibiitor

irreversible inhibition: cysteines

many enzymes have an essential cysteine in the active site. cysteines react with iodoacetamide which results in alkyation of the cysteines. once alkylated, these cysteine enzymes are irreversibly inhibited