BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 7. ENZYMOLOGY

7.6. Inhibition of Enzymatic Activity

7.6.1. Reversible Inhibition

Reversible inhibitors bind to Enzymes via weak non-covalent bonds and easily dissociate from the enzyme under certain conditions. There are two types of reversible inhibition: Competitive and non-competitive.

Competitive inhibition. This type is characterized by a reversible decrease in The rate of an enzymatic reaction caused by an inhibitor that binds to the Active Site of the enzyme and prevents The formation of the enzyme-substrate complex. This type of inhibition is observed when the inhibitor is a structural analogue of the substrate. As a result, competition arises between substrate and inhibitor molecules for the binding site within the enzyme's active site. In this case, either the substrate or the inhibitor interacts with the enzyme, forming enzyme-substrate (ES) or enzyme-inhibitor (EI) complexes. Due to the Formation of the enzyme-inhibitor complex, the reaction product is not formed (Fig. 7.21). The following equations apply to competitive inhibition:

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A classic example of competitive inhibition is the inhibition of the succinate dehydrogenase reaction by malonic acid (Fig. 7.22).

The latter is a structural analogue of succinate (possessing two carboxyl groups) and can also interact with the active site of succinate dehydrogenase. However, the removal of two hydrogen atoms from malonic acid is impossible; therefore, the reaction rate decreases.

Competitive Inhibitors reduce the rate of a chemical reaction. A competitive inhibitor increases the $K_m$ for a given substrate (decreases substrate affinity for the enzyme). This means that in the presence of a competitive inhibitor, a higher Substrate Concentration is required to reach _1/2 V_max.

Increasing The ratio of substrate to inhibitor concentration reduces the inhibitory effect. At significantly higher substrate concentrations, inhibition disappears entirely, as the active sites of all enzyme molecules will predominantly be in the complex with the substrate.

Many drugs exert their therapeutic effects through The Mechanism of competitive inhibition. For instance, quaternary ammonium bases inhibit acetylcholinesterase, which catalyzes the Hydrolysis of acetylcholine into Choline and acetic acid:

Fig. 7.21. Scheme of competitive inhibition of enzyme activity:

BS - binding site; CS - catalytic site

Fig. 7.22. Example of competitive inhibition of succinate dehydrogenase by malonic acid: I - succinate binds to the active site of the succinate dehydrogenase enzyme; II - during the enzymatic reaction, two hydrogen atoms are removed from succinate and transferred to the FAD coenzyme. As a result, Fumarate is formed and released from the active site of succinate dehydrogenase;

III - malonic acid is a structural analogue of succinate; it also binds to the active site of succinate dehydrogenase.

In this case, no chemical reaction takes place

The addition of inhibitors decreases acetylcholinesterase activity while increasing the concentration of acetylcholine (the substrate), which is accompanied by enhanced Nerve Impulse transmission. Cholinesterase inhibitors are used in the Treatment of muscular dystrophy. Effective anticholinesterase drugs include neostigmine (proserine), edrophonium, and others (Fig. 7.23).

Substances known as antimetabolites are used in medical practice as Enzyme Inhibitors acting via a competitive mechanism. Being structural analogues of natural substrates, these compounds cause competitive inhibition of enzymes on the one hand, and on the other hand, can be utilized by these same enzymes as pseudosubstrates, leading to the synthesis of abnormal products that lack functional activity. As a result, a decrease in the rate of certain metabolic pathways is observed.

Fig. 7.23. Diagram of the active site of acetylcholinesterase:

A - binding of acetylcholine at the enzyme's active site. The arrow indicates the site of ester bond hydrolysis in the acetylcholine molecule; B - binding of the competitive inhibitor neostigmine at the enzyme's active site. The site of neostigmine hydrolysis is indicated, but the reaction proceeds much slower than with acetylcholine; C - binding of the competitive inhibitor edrophonium to the enzyme's active site. Edrophonium binds at the active site of acetylcholinesterase, preventing the binding of acetylcholine

The following antimetabolites are used as medicinal drugs: sulfonamides (analogues of Para-aminobenzoic Acid) used for treating infectious diseases, and nucleotide analogues used in Cancer therapy.

Non-competitive inhibition. This term refers to an inhibition of an enzymatic reaction in which the inhibitor interacts with the enzyme at a site distinct from the active site (Fig. 7.24). Non-competitive inhibitors are not structural analogues of substrates.

Fig. 7.24. Diagram of non-competitive Enzyme Inhibition

A non-competitive inhibitor can bind to either the free enzyme or the enzyme-substrate complex, forming an inactive complex. The binding of a non-competitive inhibitor induces a conformational change in the molecule that disrupts the interaction between the substrate and the active site of the enzyme, ultimately leading to a decrease in the rate of the enzymatic reaction.

The kinetics of non-competitive inhibition is illustrated in Fig. 7.25. This type of inhibition is characterized by a decrease in the Vmax of the enzymatic reaction alongside an apparent decrease in the substrate affinity for the enzyme, which corresponds to an increase in Km.

Fig. 7.25. Effect of a non-competitive inhibitor on the enzymatic reaction rate as a function of substrate concentration: 1 - in the absence of a mixed non-competitive inhibitor; 2 - in the presence of a mixed non-competitive inhibitor; V_max - maximum reaction velocity in the absence of the inhibitor; 'V_max - maximum reaction velocity in the presence of the inhibitor; K_m - Michaelis constant in the absence of the inhibitor; ' K_m - Michaelis constant in the presence of the inhibitor