Ch 3: Mechanisms of Enzyme Inhibition Clinical Enzymology CLS 433 Saida Almashharawi 2015-2016

A. Competitive Inhibition Here inhibitor molecules are competing with the normal substrate molecules for binding to the active site of the enzyme, because the inhibitor is a structural analog of the substrate ii. Since E-I (enzyme–inhibitor complex) can react only to reform the enzyme and inhibitor, the number of enzyme molecules available for E-S formation is reduced. Suppose 100 molecules of substrate and 100 molecules of inhibitor are competing for 100 molecules of the enzyme. So, half of enzyme molecules are trapped by the inhibitor and only half the molecules are available for catalysis to form the product. iii. Since effective concentration of enzyme is reduced, the reaction velocity is decreased.

A. Competitive Inhibition (cont.) iv. In competitive inhibition, the inhibitor will be a structural analog of the substrate. There will be similarity in three dimensional structure between substrate (S) and inhibitor (I). For example, the succinate dehydrogenase reaction is inhibited by malonate (Fig. 5.19).

A. Competitive Inhibition (cont.) v. Competitive inhibition is usually reversible. Or, excess substrate abolishes the inhibition. In the previous example of 100 moles of E and 100 moles of I, if 900 moles of S are added, only 1/10th of enzyme molecules are attached to inhibitor and 90% are working with substrate. Thus 50% inhibition in the first example is now decreased to 10% inhibition (Fig. 5.20).

A. Competitive Inhibition (cont.) vi. From the graphs, it is obvious that in the case of competitive inhibition, the Km is increased in presence of competitive inhibitor. Thus competitive inhibitor apparently increases the Km. In other words, the affinity of the enzyme towards substrate is apparently decreased in presence of the inhibitor. vii. But Vmaxis not changed. Clinical significance of such inhibition is shown in Box 5.10

B. Non-competitive Inhibition (Irreversible) A variety of poisons, such as iodoacetate, heavy metal ions (lead, mercury) and oxidising agents act as irreversible non-competitive inhibitors. There is no competition between substrate and inhibitor. See Fig. 5.22.

B. Non-competitive Inhibition (Irreversible) (cont.) ii. The inhibitor usually binds to a different domain on the enzyme, other than the substrate binding site. Since these inhibitors have no structural resemblance to the substrate, an increase in the substrate concentration generally does not relieve this inhibition. The saturation curve of this type of inhibition is shown in Fig. 5.23. A comparison of the two

B. Non-competitive Inhibition (Irreversible) (cont.) types of inhibitions is shown in Table 5.6. Examples are: iia. Cyanide inhibits cytochrome oxidase. iib. Fluoride will remove magnesium and manganese ions and so will inhibit the enzyme, enolase, and consequently the glycolysis. iic. Iodoacetate would inhibit enzymes having-SH group in their active centers. iid. BAL (British Anti Lewisite; dimercaprol) is used as an antidote for heavy metal poisoning. The heavy metals act as enzyme poisons by reacting with the SH group. BAL has several SH groups with which the heavy metal ions can react and thereby their poisonous effects are reduced

B. Non-competitive Inhibition (Irreversible) (cont.) iii. The inhibitor combines with the enzymes by forming a covalent bond and then the reaction becomes irreversible The velocity (Vmax) is reduced. But Km value is not changed, because the remaining enzyme molecules have the same affinity for the substrate. iv. Increasing the substrate concentration will abolish the competitive inhibition, but will not abolish non-competitive inhibition.

C. Uncompetitive Inhibition Here inhibitor does not have any affinity for free enzyme. Inhibitor binds to enzyme–substrate complex; but not to the free enzyme. In such cases both Vmax and Km are decreased (Fig. 5.24). Inhibition of placental alkaline phosphatase (Reganiso-enzyme) by phenylalanine is an example of uncompetitiveinhibition.

D. Suicide Inhibition i. It is a special type of irreversible inhibition of enzyme activity. It is also known as mechanism based inactivation. The inhibitor makes use of the enzyme's own reaction mechanism to inactivate it (mechanism based inactivation). ii. In suicide inhibition, the structural analog is converted to a more effective inhibitor with the help of the enzyme to be inhibited. The substrate-like compound initially binds with the enzyme and the first few steps of the pathway are catalyzed. iii. This new product irreversibly binds to the enzyme and inhibits further reactions.

D. Suicide Inhibition (cont.) -. The anti-inflammatory action of Aspirin is also based on the suicide inhibition. Arachidonic acid is converted to prostaglandin by the enzyme Cyclo-oxygenase. Aspirin acetylates a serine residue in the active center of cyclo-oxygenase, thus prostaglandin synthesis is inhibited, and so inflammation subsides.

E. Allosteric Regulation Allosteric enzyme has one catalytic site where the substrate binds and another separate allosteric site where the modifier binds (allo = other) (Fig. 5.25). ii. Allosteric and substrate binding sites may or may not be physically adjacent. iii. The binding of the regulatory molecule can either enhance the activity of the enzyme (allosteric activation), or inhibit the activity of the enzyme (allosteric inhibition). iv. In the former case, the regulatory molecule is known as the positive modifier and in the latter case as the negative modifier

E. Allosteric Regulation (cont.) v. The binding of substrate to one of the subunits of the enzyme may enhance substrate binding by other subunits. This effect is said to be positive co-operativity. If the binding of substrate to one of the subunits decreases the avidity of substrate binding by other sites, the effect is called negative co-operativity. vi. In most cases, a combination is observed, resulting in a sigmoid shaped curve (Fig. 5.26). .

E. Allosteric Regulation (cont.) Salient features of allosteric regulation are enumerated in Box 5.11

F. Key enzymes Body uses allosteric enzymes for regulating metabolic pathways. Such a regulatory enzyme in a particular pathway is called the key enzyme or rate limiting enzyme. ii. The flow of the whole pathway is constrained as if there is a bottle neck at the level of the key enzyme.

F. Key enzymes iii. The allosteric inhibitor is most effective when substrate concentration is low. This is metabolically very significant. When more substrate molecules are available, there is less necessity for regulation. Two best examples are given in detail below:

G. Feedback Inhibition The term feedback inhibition or end-product inhibition means that the activity of the enzyme is inhibited by the final product of the biosynthetic pathway In this pathway, if D inhibits E1, it is called feedback inhibition. Usually such end product inhibition is effected allosterically.

Part 2 Metabolic Inhibitors

Covalent Modification The activity of enzymes may be increased or decreased by covalent modification. It means, either addition of a group to the enzyme protein by a covalent bond; or removal of a group by cleaving a covalent bond. Examples of a covalent modefication: - Zymogen activation by partial proteolysis, Addition or removal of a particular group brings about covalent modification of enzyme protein. This is a reversible reaction. 1- Commonest type is the reversible protein phosphorylation. The phosphate group may be attached to serine, threonine or tyrosine residues

Examples of a covalent modification When hormone binds to the membrane bound receptor, hormone receptor complex (HR) is formed. The enzyme adenylate cyclase is activated. This activation is mediated through a G protein. The active adenylate cyclase converts ATP to cyclic AMP (cAMP) which acts as a second messenger. It activates protein kinase, by binding to the regulatory subunit of the enzyme. The active catalytic subunit will phosphorylate the enzyme. In some cases, the receptor itself has tyrosine kinase activity which is switched on when hormone binds to receptor, e.g. insulin.

2- ADP ribosylation is another covalent modification, where an ADP-ribose from NAD+ is added to enzyme /protein. For example, ADP ribosylation of alpha subunit of G-protein leads to inhibition of GTPase activity; hence G protein remains active. - Cholera toxin and pertussis toxin act through ADP ribosylation. -ADP ribosylation of glyceraldehyde-3-phosphate dehydrogenase results in inhibition of glycolysis.

Stabilization Enzyme molecules undergo usual wear and tear and finally get degraded. Such degradation if prevented can lead to increased overall enzyme activity. This is called stabilization of enzyme. Degradation of Tryptophan pyrrolase is retarded by tryptophan. Phospho fructo kinase is stabilized by growth hormone. Enzymes having SH groups (Papain, Urease, Succinate dehydrogenase) are stabilized by glutathione (G-SH).

Compartmentalization The activity of enzymes catalysing the different steps in a metabolic pathway may be regulated by compartmentalization of enzymes. Certain enzymes of the pathway may be located in mitochondria whereas certain other enzymes of the same pathway are cytoplasmic. For example heme synthesis, urea cycle gluconeogenesis. The intermediates have to be shuttled across the mitochondrial membrane for this purpose which provides a point where controls can be exerted.

Assignment Q1: make a comparison between the following types of inhibition, and mention the effects on Vmax, Km, Substrate saturation curve, Lineweaver Burk plot (by plotting the curves) Competitive inhibitor non – Competitive inhibitor Un- Competitive inhibitor Note: your Assignment to be received before Thursday 11/Feb./2016