ENZYME CLASSIFICATIONS AND STRUCTUREpptx

Describe enzyme functions and classification

1. Enzymology Principles By George Kazibwe

2. Definitions Enzymology refers to study of enzymes Enzymes are biological catalysts that coordinate metabolism

3. Relevance of enzymology Disease diagnosis Genetic engineering Drug synthesis Food industry Wood industry Bioremediation (cleaning up the env’t using microorganisms) Plus a variety of other biological functions; Cell infection Signal transduction Active transport as ion pumps such as ATPases Digestion QN. State the rationale behind the use of enzymes as diagnostic markers.

4. Enzyme classification Most of the known enzymes are protein in nature but some require non-protein factors to become catalytically active. They can be broadly classified according to: 1. Chemical composition Simple enzymes - these are purely made up of proteins e.g. pepsin, trypsin, pancreatic ribonuclease etc Complex enzymes - are composed of the protein part and non-protein part.

5. Enzyme classification cont’d The protein part is called an apoenzyme/apoprotein whereas the non-protein part is generally referred to as a cofactor . A complete catalytically active complex enzyme is called a holoenzyme .

6. Enzyme classification cont’d A cofactor can be; A coenzyme- a non protein organic substance that is dialyzable, thermostable and loosely attached to the apoenzyme e.g. FAD + , NAD + , NADP + A prosthetic group - a non protein organic substance that is dialyzable, thermostable and firmly attached to the apoprotein e.g. heme A metal ion - a non protein inorganic substance. This can either be firmly attached ( metalloenzymes ) or its presence is required before an enzyme becomes active ( metal- activated enzymes ).

7. Role of the cofactor!

8. Cofactors & their uses

9. Enzyme classification cont’d 2. According to the chemical reaction catalyzed: The EC grouped them into six classes EC 1 Oxidoreductases : catalyze oxidation /reduction reactions. EC 2 Transferases : transfer a functional group ( e.g. a methyl or phosphate group). EC 3 Hydrolases : catalyze the hydrolysis of various bonds. EC 4 Lyases : cleave various bonds by means other than hydrolysis and oxidation. EC 5 Isomerases : catalyze isomerization changes within a single molecule. EC 6 Ligases : join two molecules with covalent bonds forming C-C, C-S, C-O & C-N.

10. Enzyme classification cont’d EC1: Oxidoreductases Oxidases- use oxygen as an electron acceptor Dehydrogenases- use molecules other than oxygen as electron acceptors Oxygenases- directly incorporate oxygen into substrates Peroxidases- use hydrogen peroxide as an electron acceptor. EC 2: Transferases Methyltransferases- transfer one carbon units between substrates Aminotransferases- transfer NH 2 from amino acids to keto acids Kinases- transfer PO 3 - from ATP to substrates Phosphorylases- transfer PO 3 - from Pi to substrates

11. Enzyme classification cont’d EC3: Hydrolases Phosphatases – remove PO 3 - from substrates e.g. Glucose-6-phosphatase Phosphodiesterases- cleave phosphodiester bonds Proteases- cleave amide bonds EC4: Lyases Decarboxylases- produce CO 2 via elimination reactions e.g. pyruvate decarboxylase Aldolases- produce aldehydes via elimination reactions EC5: Isomerases Racemases- interconvert L and D stereoisomers Mutases- transfer groups btn atoms within a molecule Epimerases- catalyze formation of epimers EC6: Ligases Carboxylases- use CO 2 as a substrate Synthases- Link two molecules via an ATP independent reaction Synthetases- link two molecules via an ATP dependent reaction

12. Enzyme specificity The specificity of an enzyme is determined by the functional groups of the substrate, the enzyme and the physical proximity of these functional groups. There are 4 distinct types of enzyme specificity: Absolute specificity - the enzyme will catalyze only one reaction. Group specificity - the enzyme will act only on molecules that have specific fxnal grps such as amino, phosphate & methyl groups. Linkage specificity - the enzyme will act on a particular type of a chemical bond regardless of the rest of the molecular structure. Stereochemical specificity - the enzyme will act on a particular steric or optical isomer.

13. Enzyme specificity: Lock & Key theory This was proposed by Emil Fischer in 1894. Stipulates that the shape of the substrate is complementary to that of the active site of the enzyme. That they are thought to fit together like a key into its lock. This was however discredited by Daniel Koshland’s induced fit theory in 1958.

14. Fischer’s Lock & Key Theory

15. Fischer’s Lock and Key theory Cont’d

16. Koshland’s Induced Fit theory This was proposed by Daniel Koshland in 1958. It stipulates that the active site in the absence of a substrate is rather a nondescript region of the enzyme. That the process of substrate binding induces specific conformational changes in the enzyme structure particularly in the region of the active site.

17. Koshland’s Induced Fit theory

18. Measures of enzyme activity Turnover number of an enzyme, k cat is the number of substrate molecules metabolized per enzyme molecule per unit time with units of per minute or per second.

19. Catalytic power of enzymes Enzymes can accelerate reactions as much as 10 16 over uncatalyzed reactions For example, Urease Catalyzed rate: 3 x 10 4 /sec Uncatalyzed rate: 3 x 10 -10 /sec

20. Enzyme catalysis

21. Enzyme catalysis cont’d Enzymes increase product formation by; Lowering the energy barrier (activation energy) for product formation Increasing the favorable orientation of colliding reactant molecules for product formation to be successful Mechanisms of enzyme catalysis: Factors that enhance the catalytic power of an enzyme Proximity and orientation effect Strain and distortion: Induced Fit General acid-base catalysis Covalent catalysis

22. Factors of catalytic power Cont’d! Proximity and orientation Catalytic groups are arranged in a such way that the probability of forming an ES complex is increased Strain and distortion Binding of a substrate induces a conformational change in the enzyme molecule which strains the structure of the active site and also distorts the bound substrate. Such a change is called the induced fit of the enzyme to the substrate. General acid-base catalysis The active site may furnish R groups of specific amino acid residues that are good proton donors or acceptors. Covalent catalysis Some enzymes form unstable, covalently joined ES complexes that breakdown into products.

23. Enzyme catalysis cont’d Examples of proton-donating groups -COOH -NH 3 + -SH Examples of proton accepting groups -COO - -NH 2 -S -

24. Enzyme kinetics Branch of enzymology that deals with the study of factors that affect velocities of enzyme catalyzed reactions. The Michaelis-Menten kinetic model is exploited which states that an enzyme catalyzed rxn involves the reversible formation of an ES, that breaks down to form E and P

25. BLT Enzymology- DAY & EVENING: 2018/2019 By Saphan

26. Significance of Km Measures the enzyme affinity for the substrate i.e. the high the Km the lower the enzyme affinity for the substrate and vice versa. Identification of physiological enzyme inhibitors. Indicates the intracellular substrate concentration. Enzyme identification and comparisons.

27. Units of velocity & substrate concentration

28. Lineweaver- Burk plot Represents a linear transformation of Michealis-Menten equation. Used to illustrate the effects of inhibitors on enzyme catalyzed reactions. Other modifications; Eadie-Hofstee plot.

29. Lineweaver-Burk Plot

30. Regulation of enzyme activities Allosteric control -regulator molecules Feedback/product inhibition Covalent modification Acetylation Phosphorylation Nucleotidylation Proteolytic activation - proenzymes /zymogens Multiple enzyme forms-LDH Regulation of enzyme activities at the gene level

31. Regulation of enzyme activities cont’d Serine regulates its rate of synthesis by acting as a feedback inhibitor

32. Regulation of enzyme activities cont’d Proteolytic events that ultimately result in formation of the catalytic site of chymotrypsin ( α- CT)

33. Factors that affect enzyme activity Substrate concentration Enzyme concentration Temperature –disrupts hydrogen bonds and hence denatures the protein pH- disrupts bonds btn amino acid residues and alters the overall charge of the protein Cofactors Inhibitors

34. Effect of pH on enzyme activity

35. Types of enzyme inhibition Divided into; Reversible enzyme inhibition Irreversible enzyme inhibition – Diisopropylfluorophosphate, OPs for acetylcholinesterase Reversible enzyme inhibition This is further divided into; Competitive e.g. malonate & oxaloacetate- succinate dehydrogenase; prostigmine & physostigmine for acetylcholinesterase Non- competitive inhibition- Isoleucine for threonine dehydratase Uncompetitive inhibition- lithium for inositol monophosphatase

36. Effect of enzyme inhibitors on K m and V max

37. Enzyme inhibitors cont’d Competitive enzyme inhibitor Binds free enzyme Structurally similar to substrate Competes with the substrate for the active site Raise Km without affecting Vmax Increasing substrate concentration relieves inhibition Non-competitive enzyme inhibitor Binds either free Enzyme or ES complex Structurally dissimilar with substrate Binds to an allosteric site Lowers Vmax and has no effect on Km because; It doesn’t bind to the active site but only alters the conformation of the enzyme to affect catalysis but not substrate binding.

38. Enzyme inhibitors cont’d Uncompetitive enzyme inhibitor Binds ES complex Prevents ES from proceeding to E + P or back to E + S Lowers Km and Vmax but the ratio of Km/Vmax remains the same Occurs with multisubstrate enzymes

39. Effect of inhibitors on K m and V max cont’d Type of inhibition Enzyme inhibitor interactions Effect on Km Vmax Competitive E + I= EI Increases None Uncompetitive ES + I= ESI Decreases Decreases Noncompetitive E + I= EI None Decreases

40. Types of enzyme inhibition cont’d Irreversible inhibitors bind covalently to the active site that the enzyme is inactivated irreversibly. Examples include Diisopropylfluorophosphate that inhibits serine proteases; idoacetate inhibits ribonuclease A; Sarin, a nerve gas that inhibits acetylcholinesterase.

41. Beware of misleading information from Internet!

42. Medical relevance of enzymology Therapeutics- the rational design of therapeutic drugs often involves the synthesis of inhibitors of certain enzymes e.g. Inhibitor Target enzyme Effect or application 5-Fluorouracil Thymidylate synthetase Antineoplastic agent Allopurinol Xanthine oxidase Treatment of gout Aspirin Cyclooxygenase Ant-inflammatory Lovastatin HMG-CoA reductase Cholesterol lowering agent Penicillin Transpeptidase Antibacterial Pargyline Monoamine oxidase Antihypertensive agent

43. Medical relevance of enzymology cont’d: Clinical Dx Clinical diagnosis - enzymes are mainly intracellularly located hence their activity in serum is always low (within the normal range). Additionally, certain enzymes are specifically produced by particular tissues thus their presence in serum occurs when a specific tissue is damaged. Therefore, measurement of enzyme activity in serum provides a clue about the tissue health status.

44. Medical relevance of enzymology cont’d: Clinical Dx Assayed enzyme Diagnostic uses: Detection of; Alanine aminotransferase Viral hepatitis, liver damage Acid phosphatase Prostate cancer Creatine kinase Muscle & heart disorders Alkaline phosphatase Liver disease, bone disorders Lactate dehydrogenase Endocarditis Amylase Acute pancreatitis