Inhibitors are molecules that affect the activity of enzymes, the control of enzyme activity through the use of inhibitors has a vast variety of uses. Inhibitors can be reversible meaning they form weak interactions with their target enzyme and are easily removed or irreversible forming strong stable interactions that can only be removed chemically. A large number of commonly used drugs are enzyme inhibitors; these include drugs to treat HIV, cancer and heart disease. Enzyme inhibitors are used in agriculture as pesticides and herbicides. Enzyme inhibitors are naturally occurring but many have also been designed and created synthetically. There are several different mechanisms of enzyme inhibition all of which have their own characteristic effect on the kinetics of the enzyme. This article will discuss the different types of inhibitors, their individual mechanisms of action and their effects on kinetics.
If we think about the steps of an enzyme catalysed reaction its clear to see at what stages inhibition can occur. An inhibitor can bind before the substrate has bound blocking the active site and preventing formation of an enzyme substrate complex, this type of inhibition is called competitive inhibition. Alternately an inhibitor can bind to the enzyme substrate complex preventing the formation or release of the product; this is known as uncompetitive inhibition.
A reversible inhibitor forms a transient interaction with the enzyme; they do not form covalent interactions but instead rely on non-covalent forces such as hydrogen bonding and electrostatic attraction. The strength of the binding between an enzyme and a reversible inhibitor is defined by the dissociation constant (Kd).
The smaller the value of Kd the stronger the interaction between the enzyme and inhibitor and the greater the inhibitory effect. When talking about enzyme inhibition Kd is referred to as Ki. Determining the value of Ki can be very useful in drug design and chemists will try to fine tune the inhibitor to achieve the optimal Ki.
As the name suggests a competitive inhibitor competes with the substrate to bind the enzyme, imagine a race in which the inhibitor and substrate are both trying to reach the enzyme first, whichever does gets to bind. The most common type of competitive inhibitors are classical competitive inhibitors which compete with the substrate to bind an enzymes active site. Because of the specificity of an enzymes active site a competitive inhibitor will often have a similar structure and chemistry to that of the enzymes natural substrate and is referred to as a substrate analogue. However unlike the substrate the inhibitor does not react when it binds the active site, it merely blocks the site preventing the substrate from binding. Non-classical competitive inhibitors do not bind to an enzymes active site; they bind a different site on the enzyme and alter the shape of the substrate binding site preventing substrate binding. Although less common non-classical competitive inhibition is of particular relevance in the control of metabolism.
The presence of inhibitors changes the kinetics of an enzymatic reaction. The type of inhibitor present can be defined by its effects on the Michaelis Menten parameters.
Km – If we think of the value of Km as a measurement affinity then we can say that the presence of a competitive inhibitor effectively lowers the enzymes affinity for its substrate. As we can see from the Michaelis Menten plot the value of km has increased, it takes a higher concentration of substrate to reach half of Vmax.
Vmax – The value of Vmax tells us the maximum velocity (rate) at which an enzyme can work in the presence of infinite substrate. The presence of a competitive inhibitor does not affect the value of Vmax, this tells us that the effect of a competitive inhibitor can be overcome by increasing substrate concentration.
One of the most widely used competitive inhibitors is statins a drug used to lower the concentration of cholesterol in the blood. Statins are competitive inhibitors of the enzyme HMG-CoA reductase an enzyme which controls the first committed step in the biosynthesis of cholesterol. Statins are structural analogues of the enzymes natural substrate HMG-CoAthey bind with high affinity to the active site of HMG-CoA reductase resulting in less cholesterol synthesis.
Lovastatin is a commonly used statin because part of the molecule is structurally similar to the enzymes natural substrate HMG-CoA.
The enzyme HIV-1 protease binds to its substrate and to a competitive inhibitor used to fight HIV. Both substrate and drug are bound to the substrate binding site. It can be useful to determine the Ki of an inhibitor, this will give an indication of the affinity the enzyme has for the inhibitor which may be useful when trying to develop clinically useful inhibitors. When dealing with a competitive inhibitor the best way to do this is by making a Dixon plot which involves recording the velocity of a reaction using a series of inhibitor concentrations and at least two different substrate concentrations.
An uncompetitive inhibitor binds only to the enzyme-substrate complex preventing the formation or release of the enzymatic products. Unlike with classical-competitive inhibition an uncompetitive inhibitor need not resemble the structure of the enzymes natural substrate. An uncompetitive inhibitor is most effective at high substrate concentration as there will be more enzyme-substrate complex for it to bind. Unlike with competitive inhibitors the effects of an uncompetitive inhibitor cannot be overcome by increasing the concentration of substrate.
Km – An uncompetitive inhibitor will reduce the value of km, however the initial slope of the curve remains the same, this is because at very low substrate concentration there is little enzyme-substrate complex for the inhibitor to bind so the effects are negligible.
Vmax – The value of Vmax is also reduced by uncompetitive inhibition. This is because the total ‘pool’ of enzymes available to react has been reduced, effectively our enzyme concentration has reduced and as Vmax is defined as Vmax = Kcat x [E] we can see why this would reduce our Vmax.
If we want to determine the Ki of an uncompetitive inhibitor then the Dixon plot is not appropriate, instead we can use a Cornish-Bowden plot, which gives us converging lines at varying substrate concentrations allowing the value of K i to be determined from the X-axis.
Mixed inhibition is a type of reversible inhibition which combines the effects of both competitive and uncompetitive inhibition. The inhibitor can bind either the enzyme or the enzyme-substrate complex and in either case will form an inactive complex. The inhibitor does not bind to the substrate binding site and therefore is not a substrate analogue. The value of Vmax will go down; the Km can go up or down depending on the individual case. Non-competitive inhibition is a special type of mixed inhibition where the value of Km is not affected by the presence of the inhibitor. Non-competitive inhibition is very rare however is occasionally seen in allosteric enzymes.
As the name suggests irreversible inhibitors permanently eradicate the activity of an enzyme. This is achieved by forming stable, often covalent, bonds with the enzyme. Typically the inhibitor will function by causing alkylation or acylation of a side chain residue of the active site. Irreversible inhibitors are often used as poisons, an example of this being the organophosphates used as insecticides, herbicides and nerve gas, which function by irreversibly inhibiting the enzyme acetyl cholinesterase. A more pleasant and familiar use of irreversible inhibition is the analgesic drug aspirin. Aspirin works by causing irreversible acylation of serine residues of the active site of COX enzymes preventing the synthesis of prostaglandins and thromboxane.
Enzyme inhibitors fall into two broad categories, irreversible and reversible. Irreversible inhibitors form strong, stable interactions with their target enzyme causing the enzyme to become permanently inactive. Irreversible inhibitors are often poisons however they are also used as therapeutics such as aspirin. Reversible inhibitors form weak, transient interactions with their target enzyme. Reversible inhibitors can be further classified according to their mechanism of action. Competitive inhibitors compete with the substrate to bind the enzyme forming an enzyme-inhibitor complex which cannot be bind substrate. Uncompetitive inhibitors bind to the enzyme-substrate complex itself. Reversible inhibitors are used extensively as therapeutic agents and also by cells to control metabolic processes. Reversible inhibitors have their own characteristic effect on the Michaelis-Menten parameters allowing the type of inhibition to be easily determined.
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