Competitive inhibition

I E I

E + S ES E + P +

I E I

On the basis of Vmax = k2.E0 relation, the presence of an irreversible inhibitor can be proven, and a distinction can be done between reversible and an irreversible inhibitors. If the maximum rate velocity is plotted against the introduced initial enzyme concentration, the picture of the Fig 2.26. will be the result. In the presence of an irreversible inhibitor, we shall get an intercept on the x-axis that gives exactly the amount of the irreversible inhibitor. This is because this inhibitor practically removes a certain part of the enzyme and those inhibitors bound enzyme molecules totally lost their activity, as if they were not present at all.

Fig 2.26.: Recognition of an irreversible inhibitor

Reversible inhibitors form dynamic complexes with the enzyme molecules, and the catalytic effect of such a bound enzyme differs from that of not inhibitor bound molecules. The types of the inhibitions may differ from the point of view that the activity is getting zero when an inhibitor molecule forms complex (this is the case of complete inhibition) or there remains a certain fraction of the original activity (= partial inhibition). The former is also called linear inhibition because the so-called Dixon-plot gives a straight line (1/V against inhibitor concentration), while the opposite case is called hyperbolic inhibition = partial inhibition. Another distinction in case of complete or linear inhibition is based upon the effect of the inhibitor on the kinetic parameters. If Vmax does not change but Km increases it is the competitive inhibition, if the inhibitor does not touch Km but decreases Vmax, we have a noncompetitive inhibitor. If both parameters decrease with a constant ratio, the inhibitor is called uncompetitive and finally if a certain mixture of effects is observed that is the case of mixed inhibition. From the scheme of the latter all the other types can be deduced. From now on we deal only with reversible linear inhibitions.

2.4.1. Competitive inhibition

Competitive inhibitors prevent the binding of a substrate molecule to the enzyme. But a previously bound substrate also prevents the binding of a competitive inhibitor to the enzyme. With other words the substrate and the inhibitor mutually exclude each other from the enzyme. This is a real competition for the binding sites of the enzyme between the substrate and the inhibitor.

Competitive inhibitors can be so called substrate analogues that are chemically similar to the substrate but are unconvertable (non-metabolizable) by the enzyme or they can be alternative substrates or products of the enzyme. These are the case of the classical competitive inhibitions. On the other hand, there exist such competitive inhibitors which are not structurally similar to the substrate but also prevent the substrate binding. Possible mechanisms of the competitive inhibition are shown on Fig 2.28. Let us observe that only the model a) corresponds a real structural similarity when the inhibitor as well as the substrate intend to bind to the same binding site.

Fig 2.27.: Models of competitive inhibition 1

Case b) is a kind of steric hindrance, here the inhibitor covers the substrate binding site and does not allow binding of the substrate (and vice versa). Model c) and d) are cases of partial or complete over-lapping of the binding sites. The situation on the Fig.2.28 is the most important from physiological point of view, because this competitive feedback inhibition is one of the enzyme level promptly acting regulation mechanisms. The end product of a series of reactions can prevent the overproduction of the given metabolite because it can combine with the enzyme of the first reaction in the series and competitively stops its conversion step thus ceasing the further unnecessary production of the given metabolite. The mechanism of this model do not need any structural similarity or close binding. The inhibitor can bind even a place far from the substrate binding site, but it causes a conformational change of the tertiary structure of the protein, that prevents the sustrate binding. All these models mean mutual exclusion either the subtrate or the inhibitor.

.

Fig 2.28.: Models of competitive inhibition 2

In order to describe the kinetics of competitive inhibition let us look at the scheme below:

E + S ES E + P hexokinase, glucose is the substrate and fructose may be an alternative substrate, the enzyme equally able to phosphorylate both.

If kap = 0, this is the case of dead-end competitive inhibition, i.e EI complex does not form any product.

Applying the mnemotechnic process we have seen in 2.3 it is possible to write a rapid equilibrium kinetics according to the scheme above.

V

representing the (EI) complex. A more familiar equation of the competitive inhibition can be got from eq (2.18) multiplying by KS and rearranging:

max

(Following the steady state way of thinking, naturally a similar equation would be the result with Km instead of KS.)

Fig.2.29.: V-S plot of competitive inhibition

The term Km(1+I/Ki) of the denominator is called Kmi apparent Michaelis-constant. As seen on the figure the Vmax does not change at competitive inhibition, I changes only the apparent Kmi value: it is increasing i.e. the inhibitor decreases the affinity of the enzyme to the substrate.

(1+I/Ki) term is worth to note because it will play role at every instance of inhibitions!

On Fig 2.30.the characteristic linearized Lineweaver–Burk plot of the competitive inhibition is shown which gives one of the graphical methods of evaluation of the parameters. It can be seen that the slope of the curves is a linear function of the inhibitor concentration, and this makes possible the Ki determination, too:

tg K

V

K

V K I

m max

m max i

α = + (2.20)

Fig 2.30.: Lineweaver–Burk plot of competitive inhibition

Let us calculate the inhibitor concentration that causes a doubling of the slope of the inhibited L-B curve.:

At a competitive inhibition Ki gives this concentration that doubles the slope of the L-B strait line.

But do not think that in this case the degree of the inhibition is 50%! Latter can be calculated this way:

S

Competitive inhibitors play an important role in chemotherapy, we know many pharmaceuticals that act as competitive inhibitors of specific important enzymes of the target cells. For some microbes (causing various contaminations in humans) p-amino-benzoic acid is a vitamin-like compound and it is showing structural similarity with several sulfonamide-drugs (Ultraseptyl, Superseptyl, Sulpha-guanidine etc).

The antibiotic cycloserine is similar to the amino acid alanine. In both cases the drug competes with the substrate of an important key enzyme of the contaminating microorganism, drastically lowers the rate of the enzymatic transformation, that would be vital for the microbes, thus kills them.

Other classical example of competitive inhibition is the succinate-dehydrogenase (EC 1.3.99.1) and its substrate tartrate and its competitive inhibitor substrate analogue malonate (Fig 2.31. ). It is interesting that the product of this enzyme - fumarate - is also a competitive inhibitor (Ki = 1,9·10^-3 Mmol).

Fig 2.31.: Sulfa drugs are competitive inhibitors

Fig 2.32.: Competitive inhibitor examples

It is not difficult to recognize the similar forms of eq (2.19) in the following analogies:

competitive product inhibition:

K S 1 P K V S V

P s

max

+



 



 +

=

Alternative or competing substrates (e.g.: hexokinase: glucose, fructose):

In document Index of /oktatas/konyvek/abet/Biology-biotechology_in_English (Pldal 45-51)