1. Mechanism of enzyme catalysis
How enzymes work

2. Enzymes
existence of catalysis in biological materials was discovered by Swedish chemist Berzelius
proteins (the first isolated enzyme was urease)
catalyze the chemical reactions necessary for life
typical increase of chemical reaction rates 1010 – times
many enzymes for their activity require cofactor called coenzyme

3. Decreasing of free energy of the transition state by catalysis
G activation free energy

4. The steps in enzyme-catalyzed reaction

5. The important effects of enzyme catalysis
proximity effect
general-base and general-acid catalysis
electrostatic effects
nucleophilic or electrophilic catalysis by functional groups of enzyme
structural flexibility

6. The proximity effect
reaction between two reactants is accelerated by holding these ones closer together in a correct orientation

7. The general-base and general-acid 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

8. The general-base and general-acid catalysis

9. Ribonuclease A - example of acid-base catalysis
small (124AA) protein that hydrolyzes RNA by cleaving ester bond between P and O of ribose carbon 5

10. Electrostatic effects
stabilization of distribution of electric charge in transition states during enzymatic reactions
the changing charges on atoms of substrate in a transition state intearacts with charges on atoms of the surrounding enzyme and also nearby water molecules

11. 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 similarly
electrophilic group of enzymes is usually its complex of metal cofactor with substrate
nucleophic catalysis involves the formation of an intermediate state in which substrate is covalently bound to a nucleophilic group

12. Nuclephilic catalysis - acetoacetic decarboxylase
an cytosolic enzyme involved in the ketone body production pathway in humans

13. Serine protease - an example of nucleophilic catalysis
serine proteases are a large family of proteolytic enzymes using this mechanism
the best known serine endoproteases are trypsin, chymotrypsin and elastase of pancreatic juice
conserved sequence , about 1/2 trypsin and chymotrypsin identical and 1/4 tr X elastase
cleavage - basic , aromatic and (less specifity) small hydrophobic(Ala)

14. Characteristics of the substrate-binding sites in chymotrypsin, trypsin and elastase
Baynes, J.W.,Dominiczak, M.H.: Medical Biochemistry

15. The mechanism of action of chymotrypsin
a) pocket for substrate, trypsin - Asp and attraction of Lys,Arg, chymotrypsin Ser - less polar for aromatics

16. 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

17. 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 measured in serum for diagnostic purposes
Typical examples of diagnostically important serum isoenzymes are CK (myocardial infarction), CGT (hepatitis) or LDH (myocardial infarction, hepatitis)
aspartate amino transferase(SGOT), creatin kinase, gama glutamyl transferase
CK enzyme consists of two subunits, which can be either B (brain type) or M (muscle type) , CK-MM, CK-BB and CK-MB
heart muscle expresses CK-MM at 70% and CK-MB at 25-30%

18. LDH isoenzymes
LDH catalyzes the interconversion of pyruvate and lactate with concomitant conversion of NADH and NAD+
tetrameric enzyme from two different subunits (H and M)
H – heart , M muscle and liver
Baynes, J.W.,Dominiczak, M.H.: Medical Biochemistry

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

20. Oxidoreductases (EC 1.)
catalyzes transfer of electrons from one molecule (reductant, electron donor) to another (oxidant, electron acceptor)
dehydrogenases catalyze oxidation reaction which involves removing hydrogen from 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.

21. 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 coenzyme
typical coenzymes of transferases are ATP, pyridixalphosphate (amino group), tetrafolic acid (formyl group), adenysylmethionine (methyl), coenzyme A (acetyl)

22. Hydrolases (EC 3.)
catalyzes the hydrolysis of a chemical bond. A–B + H2O → A–OH + B–H
cleave e.g. ester bonds (esterases, nucleases, phosphodiesterases, lipases, phosphatases), glycosidic bonds (glycosidases), peptide bonds (proteases and peptidases)
Hydrolysis is a chemical reaction during which one or more water molecules are split into hydrogen and hydroxide ions which may go on to participate in further reactions. chemical reaction

23. 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

24. 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

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

26. An enzyme catalyzed reaction proceeds rapidly under mild conditions because involved enzyme decreases the activation energy for a reaction. Enzymes are usually highly specific for the reactions they catalyze.