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 the reaction by the following equation:
Therefore, the rate acceleration provided by the catalysis can simply be calculated::
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. Transition state stabilisation

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. Mechanism for ketosteroid isomerase. Example of acid/base catalysis

12. Acid/base catalysis

13. Metal Ion catalysis
1. Metalloenzymes Tighly bound metal ions (Fe2+, Fe3+, Cu2+, Zn2+,Mn2+, Co3+) Metal-activated enzymes (Na+, K+, Mg+, Ca2+) Role of metal ions in catalysis 1) Binding of substrate and their properly orientation to reaction 2) Mediating of oxidation-reduction reactions through reversible changes in the metal ion oxidation state 3) Charge stabilization, shielding negative charges

14. Metal Ion catalysis Lactatedehydrogenase
Mediating of oxidation-reduction reactions
Lactatedehydrogenase
1) Fast bond of NAD+ in enyzmatic domain, isomerization and deprotonisation (H bonds Ser 48 and His 51)
2) Alcoholic substrate changes OH- coupled with Zn2+ reorganisation of
bonds and E-NADH- aldehyd complex formations

15. Metal Ion catalysis Mediating of oxidation-reduction reactions
3. Release of product and reduced NADH coenzyme, rearrangement

16. Metal Ion catalysis Metal Ion Enzyme Iron Cytochromes Aconitase
Catalase
Zinc
Carbonate dehydrogenase
Superoxide dismutase
Manganese
Pyruvate Carboxylase
Molybdenum
Xanthine oxidase
Copper
Cytochrome oxidase

17. Electrostatic effects
stabilization of electric charge distribution in transition states during enzymatic reactions
the changing atom charges of substrate in a transition state interacts with atom charges of the surrounding enzyme and also neighbour water molecules

18. Nucleophilic or electrophilic catalysis
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

19. Nucleophilic catalysis

20. Electrophilic catalysis

21. Nucleophilic catalysis - acetoacetic decarboxylase

22. 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

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

24. The mechanism of chymotrypsin action

25. 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

26. Enzyme flexibility – the use of strain energy

27. 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)

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

29. Classifying enzymes (1972 International Union of Biochemistry)
Oxidoreductases
Transferases
Hydrolases
Lyases
Isomerases
Ligases (synthetases)

30. Oxidoreductases (EC 1.)
catalyze transfer of electrons from one molecule (reductant, electron donor) to another (oxidant, electron acceptor)
dehydrogenases catalyze oxidation reaction which involves removing hydrogen from the reductant
typical coenzymes are nicotine nucleotides (NADH, NADPH), flavin nucleotides (FMN, FAD), hemins, coenzyme Q (ubichinone) and lipoic acid
typical representants are alcohol dehydrogenase, glucosooxidase etc.

31. 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)

32. Hydrolases (EC 3.) catalyzes the hydrolysis of a chemical bond:
A–B + H2O → A–OH + B–H
cleave, for instance, ester bonds (esterases, nucleases, phosphodiesterases, lipases, phosphatases), glycosidic bonds (glycosidases), peptide bonds (proteases and peptidases)

33. Lyases (EC 4.)
cleave C-C, C-O, C-N and other bonds by other means than by hydrolysis or oxidation
require only one substrate for the reaction in one direction, but two substrates for the reverse reaction (e.g. adenyl cyclase catalyzes ATP → cAMP + PPi )
decarboxylases (EC 4.1.1) are lyases cleaving C-C bond and liberates carbon dioxide from carboxyl group

34. Isomerases (EC 5.)
catalyze reactions involving a structural rearrangement of a molecule
e.g. alanine racemase 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

35. Ligases (synthetases) (EC 6.)
catalyze synthesis of a new bond between two molecules
reaction is usually accompanied by hydrolysis of ATP or another similar triphosphate
biotin is a cofactor for enzymes catalyzing carboxylation binding carbon dioxide to molecule) called carboxylases (e.g. pyruvate carboxylase)

36. Literature
Baynes, J.W.,Dominiczak, M.H.: Medical Biochemistry, Elsevier 2004
Bugg, T.:Introduction to Enzyme and Coenzyme Chemistry, Blackwell Publishing, 2004