1. Introduction. Structure, properties and biological functions of proteins. Methods of secretion and purification. Peptides. Complex proteins, their biological role. Structure and physical-chemical properties of enzymes

2. Structure of enzymes Enzymes
Complex or holoenzymes (protein part and nonprotein part – cofactor)
Simple (only protein)
Apoenzyme (protein part)
Cofactor
Prosthetic groups
usually small inorganic molecule or atom;
usually tightly bound to apoenzyme
Coenzyme
-large organic molecule
-loosely bound to apoenzyme

3. Example of prosthetic group
Example of metalloenzyme: carbonic anhydrase contains zinc
Metalloenzymes contain firmly bound metal ions at the enzyme active sites (examples: iron, zinc, copper, cobalt).

4. Coenzyme classification
Coenzymes
Coenzymes act as group-transfer reagents
Hydrogen, electrons, or groups of atoms can be transferred
Coenzyme classification
(1) Metabolite coenzymes - synthesized from common metabolites
Vitamin-derived coenzymes - derivatives of vitamins
Vitamins cannot be synthesized by mammals, but must be obtained as nutrients

5. Examples of metabolite coenzymes
ATP can donate phosphoryl group
ATP
S-adenosylmethionine
donates methyl groups in many biosynthesis reactions
S-adenosylmethionine

6. 5,6,7,8 - Tetrahydrobiopterin
Cofactor of nitric oxide synthase

7. Vitamin-Derived Coenzymes
Vitamins are required for coenzyme synthesis and must be obtained from nutrients
Most vitamins must be enzymatically transformed to the coenzyme
Deficit of vitamin and as result correspondent coenzyme results in the disease

8. NAD+ and NADP+
Nicotinic acid (niacin) an nicotinamide are precursor of NAD and NADP
Lack of niacin causes the disease pellagra
NAD and NADP are coenzymes for dehydro-genases

9. FAD and FMN
Flavin adenine dinucleotide (FAD) and Flavin mononucleotide (FMN) are derived from riboflavin (Vit B2)
Flavin coenzymes are involved in oxidation-reduction reactions
FMN (black), FAD (black/blue)

10. Thiamine Pyrophosphate (TPP)
TPP is a derivative of thiamine (Vit B1)
TPP participates in reactions of: (1) Oxidative decarboxylation (2) Transketo-lase enzyme reactions

11. Pyridoxal Phosphate (PLP)
PLP is derived from Vit B6 family of vitamins
PLP is a coenzyme for enzymes catalyzing reactions involving amino acid metabolism (isomerizations, decarboxylations, transamination)

12. Enzymes active sites Substrate usually is relatively small molecule
Enzyme is large protein molecule
Therefore substrate binds to specific area on the enzyme
Active site – specific region in the enzyme to which substrate molecule is bound

13. Active site of lysozym consists of six amino acid residues which are far apart in sequence

14. Active site contains functional groups (-OH, -NH, -COO etc)
Binds substrates through multiple weak interactions (noncovalent bonds)

15. Theories of active site-substrate interaction
Fischer theory (lock and key model)
The enzyme active site (lock) is able to accept only a specific type of substrate (key)

16. Koshland theory (induced-fit model)
The process of substrate binding induces specific conformational changes in the the active site region

17. Specificity of enzymes
Properties of Enzymes
Specificity of enzymes
Absolute – one enzyme acts only on one substrate (example: urease decomposes only urea; arginase splits only arginine)
Relative – one enzyme acts on different substrates which have the same bond type (example: pepsin splits different proteins)
Stereospecificity – some enzymes can catalyze the transformation only substrates which are in certain geometrical configuration, cis- or trans-

18. Sensitivity to pH
Each enzyme has maximum activity at a particular pH (optimum pH)
For most enzymes the optimum pH is ~7 (there are exceptions)

19. Sensitivity to temperature
Each enzyme has maximum activity at a particular temperature (optimum temperature)
-Enzyme will denature above 45-50oC
-Most enzymes have temperature optimum of 37o

20. Naming of Enzymes Common names
are formed by adding the suffix –ase to the name of substrate
Example: tyrosinase catalyzes oxidation of tyrosine; cellulase catalyzes the hydrolysis of cellulose
Common names don’t describe the chemistry of the reaction
Trivial names
Example: pepsin, catalase, trypsin.
Don’t give information about the substrate, product or chemistry of the reaction

21. The Six Classes of Enzymes
1. Oxidoreductases
Catalyze oxidation-reduction reactions
- oxidases peroxidases dehydrogenases

22. 2. Transferases
Catalyze group transfer reactions

23. 3. Hydrolases
Catalyze hydrolysis reactions where water is the acceptor of the transferred group
- esterases peptidases glycosidases

24. 4. Lyases
Catalyze lysis of a substrate, generating a double bond in a nonhydrolytic, nonoxidative elimination

25. 5. Isomerases
Catalyze isomerization reactions

26. 6. Ligases (synthetases)
Catalyze ligation, or joining of two substrates
Require chemical energy (e.g. ATP)

27. Kinetic properties of enzymes
Study of the effect of substrate concentration on the rate of reaction

28. Enzyme inhibition
In a tissue and cell different chemical agents (metabolites, substrate analogs, toxins, drugs, metal complexes etc) can inhibit the enzyme activity
Inhibitor (I) binds to an enzyme and prevents the formation of ES complex or breakdown it to E + P

29. Reversible and irreversible inhibitors
Reversible inhibitors – after combining with enzyme (EI complex is formed) can rapidly dissociate
Enzyme is inactive only when bound to inhibitor
EI complex is held together by weak, noncovalent interaction
Three basic types of reversible inhibition: Competitive, Uncompetitive, Noncompetitive

30. Reversible inhibition Competitive inhibition
•Inhibitor has a structure similar to the substrate thus can bind to the same active site
•The enzyme cannot differentiate between the two compounds
•When inhibitor binds, prevents the substrate from binding
•Inhibitor can be released by increasing substrate concentration

31. Example of competitive inhibition
Benzamidine competes with arginine for binding to trypsin

32. Noncompetitive inhibition
• Binds to an enzyme site different from the active site
• Inhibitor and substrate can bind enzyme at the same time
•Cannot be overcome by increasing the substrate concentration

33. Uncompetitive inhibition
Uncompetitive inhibitors bind to ES not to free E
This type of inhibition usually only occurs in multisubstrate reactions

34. Irreversible Enzyme Inhibition Irreversible inhibitors
very slow dissociation of EI complex
Tightly bound through covalent or noncovalent interactions
Irreversible inhibitors
•group-specific reagents
•substrate analogs
•suicide inhibitors

35. Group-specific reagents
–react with specific R groups of amino acids

36. Substrate analogs
–structurally similar to the substrate for the enzyme
-covalently modify active site residues

37. Suicide inhibitors
•Inhibitor binds as a substrate and is initially processed by the normal catalytic mechanism
•It then generates a chemically reactive intermediate that inactivates the enzyme through covalent modification
•Suicide because enzyme participates in its own irreversible inhibition

40. Regulation of enzyme activity Methods of regulation of enzyme activity
• Allosteric control
• Reversible covalent modification
• Isozymes (isoenzymes)
• Proteolytic activation

41. Allosteric enzymes
Allosteric enzymes have a second regulatory site (allosteric site) distinct from the active site
Allosteric enzymes contain more than one polypeptide chain (have quaternary structure).
Allosteric modulators bind noncovalently to allosteric site and regulate enzyme activity via conformational changes

42. Example of allosteric enzyme - phosphofructokinase-1
(PFK-1)
PFK-1 catalyzes an early step in glycolysis
Phosphoenol pyruvate (PEP), an intermediate near the end of the pathway is an allosteric inhibitor of PFK-1
PEP

43. Phosphorylation reaction

44. Dephosphorylation reaction
Usually phosphorylated enzymes are active, but there are exceptions (glycogen synthase)
Enzymes taking part in phospho-rylation are called protein kinases
Enzymes taking part in dephosphorylation are called phosphatases

45. Isoenzymes (isozymes)
Some metabolic processes are regulated by enzymes that exist in different molecular forms - isoenzymes
Isoenzymes - multiple forms of an enzyme which differ in amino acid sequence but catalyze the same reaction
Isoenzymes can differ in:
kinetics,
regulatory properties,
the form of coenzyme they prefer and
distribution in cell and tissues
Isoenzymes are coded by different genes

46. Example: lactate dehydrogenase (LDG)
Lactate + NAD pyruvate + NADH + H+
Lactate dehydrogenase – tetramer (four subunits) composed of two types of polypeptide chains, M and H
There are 5 Isozymes of LDG:
H4 – heart
HM3
H2M2
H3M
M4 – liver, muscle
• H4: highest affinity; best in aerobic environment
•M4: lowest affinity; best in anaerobic environment
Isoenzymes are important for diagnosis of different diseases

47. Examples of specific proteolysis
Activation by proteolytic cleavage
• Many enzymes are synthesized as inactive precursors (zymogens) that are activated by proteolytic cleavage
• Proteolytic activation only occurs once in the life of an enzyme molecule
Examples of specific proteolysis
•Digestive enzymes
–Synthesized as zymogens in stomach and pancreas
•Blood clotting enzymes
–Cascade of proteolytic activations
•Protein hormones
–Proinsulin to insulin by removal of a peptide