Basics of Enzyme Catalysis

Basics of Enzyme Catalysis

1. ENZYME CATALYSIS Department of Pharmacy University of Peshawar

2. Contents Enzyme catalysis Mechanisms of enzyme catalysis Enzyme inhibition Types and mechanisms of enzyme inhibition

3. Definition Catalysis “An increase in the rate of reaction due to participation of a catalyst”. Enzyme Catalysis It is a sub-type of catalysis and is defined as “an increase in the rate of a chemical reaction due to the participation of an enzyme”.

4. Characteristics of Enzyme Catalysis The main characteristics of enzymatic catalysis include: Efficiency Enzymes are highly efficient, one molecule of an enzyme can transform more than one million molecules of substrate a minute. Specificity Enzymes are highly selective and produce only one type of product Activity Enzymes are highly active under optimum temperature and pH

5. Classification of Enzyme Based on their function enzymes are classified in to six classes Oxidoreductases Catalyzes oxidation-reduction reactions by the transfer of electrons as addition or removal of hydrogen atoms e.g. Cytochrome P450 Transferases Catalyzes transfer of group usually attached to a co-factor e.g. Hexokinase, Methyl transferase Hydrolases Catalyzes hydrolysis reactions i.e. transfer of functional groups of water e.g. Chymotrypsin. Lyases Catalyzes the addition of a functional group by breaking a double bond or vice versa e.g. Deaminase Isomerases Catalyzes the transfer of functional groups within a molecule forming isomers e.g. Phosphoglucose isomerase. Ligases Catalyzes the formation of a C-C, C-S, C-N, C-O bonds due to condensation reaction e.g. DNA ligase

6. Process of Enzyme Catalysis Enzymes achieve their enormous reaction rate accelerations by reducing the activation energy via the same catalytic mechanisms used by chemical catalysts.

7. Mechanisms of Enzyme Catalysis Enzymes carryout their function by the following mechanisms: Acid-Base Catalysis Covalent Bond Catalysis Metal-Ion Catalysis Proximity & Orientation Based Catalysis

8. Acid-Base Catalysis A proton transfer from an acid or hydroxyl from a base lowers the activation energy of a reaction Ionizable functional groups of amino acyl side chains and of prosthetic groups contribute to catalysis by acting as acid or base The catalytic activity is pH sensitive Functional groups of amino acids such as glutamic acid, aspartic acid, lysine, histidine act as an acid or base. Example Bovine pancreatic RNase A is an acid-base catalyst, it hydrolyzes RNA to its component nucleotides in the intestine.

9. Covalent Bond Catalysis Catalysis is achieved by the transient formation of a covalent bond between the enzyme and substrate. Usually the covalent bond is formed by the reaction of a nucleophilic group on the catalyst with an electrophilic group on the substrate. Therefore, it is also known as nucleophilic catalysis. The following functional group may form a covalent bond. Amino group of Lysine Imidazole group of Histidine Carboxyl group of Aspartic acid Hydroxyl group of Serine Examples Acetylcholine esterase, Chymotrypsin, Trypsin, elastase and alkaline phosphatase

10. Mechanism

11. Metal-Ion Catalysis More than 30% of all enzymes require a metal ion for its catalysis. Such enzymes are also known as metalloenzymes. Most common metal ions include Fe 2 + , Fe 3 + , Cu 2 + , Mn 2 + , Co 2 + . Catalysis by metalloenzymes takes place by the following three mechanism Binding to substrate and orient them properly for reaction. Mediating oxidation-reduction reaction through reversible changes in metal ion oxidation state. Electrostatically stabilizing or shielding negative charges. Examples: Carbonic anhydrase (Zn), Cytochrome P450 (Fe)

12. Mechanism

13. Proximity & Orientation Based Catalysis Enzyme substrate interaction align the reactive chemical groups and hold them close together in optimal geometry thus increasing the rate of reaction. This reduces the entropy of the reactants thus making addition or transfer favorable. Example: Nucleotide monophosphate kinase brings 2 nucleotides close together and facilitate the transfer of a phosphoryl group.

14. Proximity & Orientation Based Catalysis

15. Enzyme Inhibition Enzyme inhibition is process of reducing or abolishing the catalytic activity of an enzyme due to the interaction of an enzyme inhibitor. Enzyme Inhibitor They are low molecular weight compounds that bind to an enzyme and inhibit or reduce its activity by either preventing the formation or breakdown of enzyme substrate complex.

16. Types of Enzyme Inhibition

17. Reversible inhibition Reversible inhibitors bind to enzyme with noncovalent interactions, such as hydrogen bonds, ionic bonds, and hydrophobic interactions. Can be rapidly dissociated Can be readily removed by dialysis Fully active enzyme can be recovered

18. Competitive Inhibition A Competitive inhibitor is any compound which closely resembles the chemical structure and geometry of the substrate for an enzyme. The inhibitor competes for the same active site as the substrate molecule. The inhibitor interacts with the active site of the enzyme but no interaction takes place. Because of the presence of inhibitor fewer sites are available for substrate. Since the enzyme's overall structure is unaffected by the inhibitor, it is still able to catalyze the reaction on substrate molecules that do bind to an active site. Since the inhibitor and substrate bind at the same site, competitive inhibition can be overcome simply by raising the substrate concentration. Therefore, the amount of enzyme inhibition depends upon the inhibitor concentration, substrate concentration, and the relative affinities of the inhibitor and substrate for the active site. Its inhibitory action is proportional to concentration.

19. Mechanism of Competitive Inhibitors Examples: ACEIs , Statins

20. Non-Competitive Inhibitors The inhibitor usually binds non-covalently to a domain on the enzyme other than the substrate binding site. This occurs when the substances do not resemble the geometry of the substrate & do not exhibit mutual competition. They are not influenced by concentration of the substrate. Strong affinity for inhibitors prevent catalysis possibly due to distortion in enzyme conformation.

21. Examples of Non-Competitive Inhibitors Cytochrome C oxidase (Fe) inhibition by cyanide, Organophosphates bind with the serine residue to acetyl choline esterases , MAOIs , .

22. Un-Competitive Inhibitors Inhibitor has no affinity for the enzyme free enzyme. Inhibitor binds to enzyme substrate complex. Example: Arsenic inhibition of Glyceraldehyde 3-phosphate dehydrogenase (Glycolysis), Lithium inhibition of inositol monophosphate.

23. Irreversible Inhibition Destroys a functional group necessary for the catalytic action. The catalytic activity of enzyme is completely lost. The enzyme can only be restored by synthesizing new molecules. Enzyme bonds covalently with the inhibitor. It includes: Suicide inhibition

24. Mechanism

25. Suicide Inhibition It is a specialized type of irreversible inhibition (aka. Mechanism based inhibition). The structural analogue is converted into a more effective inhibitor due to enzymatic action. The substrate like compound initially binds with the enzyme and the first few steps of the pathway are catalyzed. This new product irreversibly binds to the enzyme and inhibits further reactions. Example: Allopurinol forms alloxathine which permanently inhibits xanthine oxidase, 5-fluorouracil forms 5- fluorodeoxyuridylate which inhibitis thymidylate synthase, Organophosphates with Ach.

26. Allosteric Inhibition Allosteric means other site. When an inhibitor binds to the allosteric site it induces a conformational change in the structure of the enzyme, which results in distorted shape of the active site, hence the substrate can’t bind to it. The inhibitor is not a substrate analogue. It is partially reversible when excess substrate is added. Example: Citric acid inhibiting blood clotting.

27. Mechanism

28. Michaelis – Menten kinetics It is named after German biochemist Leonor Michaelis and Canadian physician Maud Menten . The model takes the form of an equation describing the rate of enzymatic reactions, by relating reaction rate to the concentration of a substrate. The Michaelis-Menten equation has been used to predict the rate of product formation in enzymatic reactions for more than a century. Vo= Rate of reaction Vmax = Maximum reaction rate [S]= Substrate concentration KM= Michaelis constant

29. Michaelis – Menten Plot

30. Michaelis – Menten Plot

31. Lineweaver -Burk Kinetics The Lineweaver –Burk plot (double reciprocal plot) is a graphical representation of the Lineweaver –Burk equation of enzyme kinetics. It was first described by Hans Lineweaver and Dean Burk in 1934. The plot provides a useful graphical method for analysis of the Michaelis – Menten equation. The Lineweaver –Burk plot was widely used to determine important terms in enzyme kinetics, such as Km and Vmax , before the wide availability of powerful computers and non-linear regression software.

32. Lineweaver -Burk Kinetics The y-intercept of such a graph is equivalent to the inverse of Vmax . The x-intercept of the graph represents −1/Km. It also gives a quick, visual impression of the different forms of enzyme inhibition.

33. Lineweaver -Burk Kinetics When used for determining the type of enzyme inhibition, the Lineweaver –Burk plot can distinguish competitive, non-competitive and uncompetitive inhibitors. Competitive inhibitors have the same y-intercept as uninhibited enzyme (since Vmax is unaffected by competitive inhibitors the inverse of Vmax also doesn't change) but there are different slopes and x-intercepts between the two data sets. Non-competitive inhibition produces plots with the same x-intercept as uninhibited enzyme (Km is unaffected) but different slopes and y-intercepts. Uncompetitive inhibition causes different intercepts on both the y- and x-axes .

34. Lineweaver -Burk Plot

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