Non-competitive inhibition

A non-competitive inhibitor does not have influence onto the binding of the substrate and vice versa.

I and S incidentally, reversibly and independently bind to different binding sites of the enzyme molecule, i.e I and E produce complex (EI), and S and E complex (ES), but at the same time a terner (ESI) complex may also be established as the next scheme shows:.

K

from where, with a bit rearrangement – expressing every complex with E and Ks or Ki, we get the rate equation of the competitive inhibition:

V

Introducing Vmaxi apparent maximum velocity, (2.23) can be rewritten as follows:

V V S

K S ahol V V 1

1 I

K

maxi

s maxi max

i

= + =

+

In this case inhibitor changes the value of maximum velocity while does not change the value of KS (or Km).This means, that inhibitor binds to another binding site and does not influence the binding of the substrate – does not change the affinity of the enzyme to the substrate. It is important that classic noncompetitive inhibition exist only in the case of rapid equilibrium, i.e. KS=Km.

On the next Fig 2.33 characteristic plots of noncompetitive inhibition are presented.

Fig 2.33: M-M and L-B plots of noncompetitive inhibition

Slope of the L-B strait line here is a similar linear function of the inhibitor concentration as in the competitive case, thus plotting the equation gives the same curve.

As an example of the noncompetitive inhibition, see the effect of H⁺ ions onto chymotrypsin. Here in the active site, there is a proton acceptor place, which can be inhibited by increasing proton concentration. A L-B plot proves a pure noncompetitive inhibition but remember also to the complex activity influencing effect of pH.

Other examples are the heavy metals (SH-reagents) or the cyanides. With these – as we already have seen – there are always irreversible inhibitory effects, too.

Distinction can be done between a competitive and a noncompetitive inhibitor comparing the L-B plots (or of course the other linearization methods) as the Fig 2.34. shows.

Browning of apple slices on air is caused by a catechol-oxidase (this is an o-diphenol oxidase) enzyme that oxidases catechol to o-chinon. (A similar reaction is catalyzed by tyrosinase that converts tyrosine to melanin.) A competitive inhibitor of this enzyme is the substrate analogue p-hydroxy-benzoic acid while its noncompetitive inhibitor is the phenyl-thiourea. The very different kinetic behavior of these two can assumingly follow in Fig 2.34.

Fig 2.34.: Comp. and noncomp. inhibition of catechol-oxidase 2.4.3. Uncompetitíve inhibítion

An uncompetitive inhibitor is not able to bind to the free enzyme, merely to the formerly substrate-bound one. Thus an inactive (ESI) complex is formed, from which product does not releases. The simplified scheme is the following:

K

It strikes one’s eye that even infinitely high S concentration cannot stop the effect of I – always there will be – depending on I and KI – nonproductive (ESI) complex. This can be imagined that on the original free enzyme there is no such a domain which is able to accept an inhibitor molecule, an inhibitor binding site is getting formed by a conformational change caused by a substrate molecule binding process (induced fit). But at the same time the active site also undergoes a conformational change, making (ESI) complex not able to form product anymore. Moreover, this ternary complex is more stable than the simple (ES) complex.

The previously good working method here also can be followed, and the resulted kinetic equation is:

V

The Briggs–Haldane approach gives the same form, with Km. Unfortunately from this equation we cannot see whether Vmax and/or Km have been changed. To make this clear, two step rearrangement is necessary: inhibition: while the effect on Vmax is the same as in the case of noncompetitive inhibition but a reverse effect came in the case of Km. Apparent Km decreases. This shown on Fig 2.35., an uncompetitive inhibitor decreases both, Vmax and apparent Km by the same factor.

Fig 2.35.: Uncompetitive inhibition

Uncompetitive inhibitors have enormous effect on enzymatic reactions, moreover this effect increases with increasing substrate concentrations. This fact could be the explanation why uncompetitive inhibitors are so rare in the nature (contrary to competitive) and at the same time artificial such compounds are why so efficacious.

A good example is the Glyphosate (Roundup) [N-(phospho-methyl)-glycine (Fig 2.36.)], the well-known herbicide. It is an uncompetitive inhibitor of the 5-enol-pyruvil-shikimate-3-phosphate synthase [(ESPS)-synthase] which plays an important role in the synthesis of aromatic amino acids.

Along the inhibited reaction 5-enolpyruvil-shikimate-3P, a precursor of the essential chorismic acid is not produced consequently the plant will not be able to produce aromatic amino acids.

Fig 2.36.: Glyphosate is an uncompetitive inhibitor 2.4.4. Mixed inhibition

The next scheme shows the mechanism of the mixed inhibition, which is a special case of the noncompetitive inhibition. (As a matter of fact, the opposite is right!)

E + S ES E + P

According to the scheme, presence of the inhibitor modifies the dissociation of the substrate from the enzyme, that is why the effective Ks for the step EI+S is αKS.The same reason modifies the dissociation constant of ESI to αKi. This means, with other words, that the equilibrium constant of the overall reaction E ES ESI and E EI ESI is independent upon the way of the reaction,

With the ordinary method of deduction as well as applying the mnemotechnical aid we can get the kinetic equation (2.26). Here both, KS, and Vmax are modified as a function of the inhibitor concentration. Characteristic plots are in Fig.2.37.

Fig 2.37: Mixed inhibition (1<α<∞ ^{) (KS= Km)}

Looking deeply into the starting scheme, even all the submitted types of inhibitions would have been deduced from that scheme. Some textbooks follow that way.

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