Enzymology . How enzymes work - mechanisms.

1. Enzymology. How enzymes work - mechanisms. Bruno Sopko

2. A thermodynamic model of catalysis

3. A thermodynamic model of catalysis • The rate of a chemical reaction is related to the activation energy of thereaction by the following equation: • Therefore, the rate acceleration provided by the catalysis can simply becalculated:: If, for example, a catalyst can provide 10 kJ/mol1 of transition stabilisation energy for a reaction at 25º C a 55-fold rate acceleration will result, whereas a 20 kJ/mol stabilisation will give a 3000-fold acceleration and a 40 kJ/mol stabilisation a 107-fold acceleration!

4. A thermodynamic model of catalysis

5. The steps in enzyme-catalyzed reaction

6. The important effects of enzyme catalysis • proximity effect • transition state stabilisation • acid/base catalysis • electrostatic effects • nucleophilic or electrophilic catalysis by functional groups of enzyme • structural flexibility

7. Proximity effect

8. Transitionstatestabilisation

9. Acid/base catalysis • this catalysis avoids the need of extremely low or high pH • principle is to make a potentially reactive group more reactive by increasing their nucleophilic or electrophilic character by adding or removing a proton

10. Acid/base catalysis

11. Mechanismforketosteroidisomerase. Exampleofacid/base catalysis

12. Electrostaticeffects • stabilization of electric charge distribution in transition states during enzymatic reactions • the changing atom charges of substrate in a transition state interacts with atomcharges of the surrounding enzyme and also neighbourwater molecules

13. Nucleophilicorelectrophiliccatalysis • enzymatic functional groups provide nucleophilic and electrophilic catalysts • typical nucleophilic groups are amino, hydroxyl and thiol groups of AA residues but imidazol group of His or carboxyl group of Asp, Glu can serve as well • electrophilic group of enzymes is usually complex of metal cofactor with substrate • nucleophilic catalysis involves the formation of an intermediate state in which substrate is covalently bound to a nucleophilic group

14. Nuclephiliccatalysis - acetoaceticdecarboxylase

15. Serine proteases - examples of nucleophilic catalysis • serine proteases belong to large family of proteolytic enzymes using this mechanism • the best known serine endoproteases are trypsin, chymotrypsin and elastase of pancreatic juice

16. Characteristics of the substrate-binding sites in chymotrypsin, trypsin and elastase

17. The mechanism of chymotrypsin action

18. Hexokinase - example of structural flexibility increasing the specifity of enzymes Hexokinase catalyzes the transfer of phosphate group from ATP to glucose: ATP + Glc→ ADP + Glc-6-phosphate

19. Enzyme flexibility – the use of strain energy

20. Isoenzymes • Isoenzymes are enzymes that catalyse the same reaction, but differ in their primary structure and/or subunit composition • Amounts of some tissue-specific enzymes are determined in serum for diagnostic purposes • Typical examples of diagnostically important serum isoenzymes are CK (myocardial infarction), GGT (hepatitis) or LDH (myocardial infarction, hepatitis)

21. LDH isoenzymes • LDH catalyzes the interconversion of pyruvate and lactate with accompanying conversion of NADH and NAD+ • tetrameric enzyme madeof two different subunits (H and M)

22. Classifyingenzymes(1972 International Union ofBiochemistry) • Oxidoreductases • Transferases • Hydrolases • Lyases • Isomerases • Ligases (synthetases)

23. Oxidoreductases (EC 1.) • catalyze transfer ofelectronsfromonemolecule (reductant, electron donor) to another (oxidant, electronacceptor) • dehydrogenasescatalyzeoxidationreactionwhichinvolvesremovinghydrogenfromthereductant • typicalcoenzymes are nicotinenucleotides (NADH, NADPH), flavin nucleotides (FMN, FAD), hemins, coenzyme Q (ubichinone) andlipoicacid • typicalrepresentants are alcoholdehydrogenase, glucosooxidaseetc.

24. Transferases (EC 2.) • catalyze the transfer of a functional group (e.g. methyl, acyl, phospho, glycosyl etc.) from one molecule (donor) to another (acceptor) • donor molecule is often a coenzyme • typical coenzymes of transferases are ATP, pyridixalphosphate (amino group), tetrafolic acid (formyl group), adenysylmethionine (methyl), coenzyme A (acetyl)

25. Hydrolases (EC 3.) • catalyzes the hydrolysis of a chemical bond: A–B + H2O → A–OH + B–H • cleave, for instance, esterbonds (esterases,nucleases, phosphodiesterases, lipases, phosphatases),glycosidicbonds (glycosidases), peptide bonds (proteases andpeptidases)

26. Lyases (EC 4.) • cleave C-C, C-O, C-N and other bonds by other means than by hydrolysis or oxidation • require onlyone substratefor the reaction in one direction, but two substrates for the reverse reaction (e.g. adenylcyclasecatalyzesATP → cAMP + PPi) • decarboxylases (EC 4.1.1)are lyasescleaving C-C bond andliberatescarbon dioxide fromcarboxylgroup

27. Isomerases (EC 5.) • catalyze reactions involving a structural rearrangement of a molecule • e.g.alanineracemase catalyzes the conversion of L-alanine into its isomeric (mirror-image) form, D-alanine • isomerase called mutarotase catalyzes the conversion of a-D-glucose into b-D-glucose. • UDP-Glc-epimerase : UDP-Glc ⇌UDP-Gal

28. Ligases (synthetases) (EC 6.) • catalyzesynthesisof a new bond betweentwomolecules • reactionisusuallyaccompanied by hydrolysisof ATP oranothersimilartriphosphate • biotin is a cofactorforenzymescatalyzingcarboxylationbinding carbon dioxide to molecule) calledcarboxylases (e.g. pyruvatecarboxylase)

29. Literature • Baynes, J.W.,Dominiczak, M.H.: Medical Biochemistry, Elsevier 2004 • Bugg, T.:Introduction to Enzyme and • Coenzyme Chemistry, BlackwellPublishing, 2004