1. Biochemistry - as science. Structure and properties of enzymes. The mechanism of enzymes activity. Isoenzymes. Classification of enzymes. Basic principles of metabolism. Common pathways of proteins, carbohydrates and lipids transformation.

2. 1. Catalyze only thermodynamically possible reactions 2. Are not used or changed during the reaction. 3. Don’t change the position of equilibrium and direction of the reaction 4. Usually act by forming a transient complex with the reactant, thus stabilizing the transition state Common features for enzymes and inorganic catalysts:

3. 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

4. Specific features of enzymes: 1. Accelerate reactions in much higher degree than inorganic catalysts 2. Specificity of action 3. Sensitivity to temperature 4. Sensitivity to pH Metalloenzymes contain firmly bound metal ions at the enzyme active sites (examples: iron, zinc, copper, cobalt). Example of metalloenzyme: carbonic anhydrase contains zinc Example of prosthetic group

5. 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 (2) Vitamin-derived coenzymes - derivatives of vitamins Vitamins cannot be synthesized by mammals, but must be obtained as nutrients

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

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

8. 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

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

10. FAD and FMN Flavin adenine dinucleotide (FAD) and Flavin mononucleotide (FMN) are derived from riboflavin (Vit B 2 ) Flavin coenzymes are involved in oxidation-reduction reactions FMN (black), FAD (black/blue)

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

12. Pyridoxal Phosphate (PLP) PLP is derived from Vit B 6 family of vitamins PLP is a coenzyme for enzymes catalyzing reactions involving amino acid metabolism (isomerizations, decarboxylations, transamination)

13. Pyridoxal Phosphate (PLP) PLP is derived from Vit B 6 family of vitamins PLP is a coenzyme for enzymes catalyzing reactions involving amino acid metabolism (isomerizations, decarboxylations, transamination)

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

15. Characteristics of active sites  Specificity (absolute, relative (group), stereospecificity)  Small three dimensional region of the protein. Substrate interacts with only three to five amino acid residues. Residues can be far apart in sequence  Binds substrates through multiple weak interactions (noncovalent bonds)  There are contact and catalytic regions in the active site

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

17. 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)

18. Properties of Enzymes Specificity of enzymes 1.Absolute – one enzyme acts only on one substrate (example: urease decomposes only urea; arginase splits only arginine) 2.Relative – one enzyme acts on different substrates which have the same bond type (example: pepsin splits different proteins) 3.Stereospecificity – some enzymes can catalyze the transformation only substrates which are in certain geometrical configuration, cis- or trans-

19. 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)

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

21. 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

22. Principle of the international classification All enzymes are classified into six categories according to the type of reaction they catalyze Each enzyme has an official international name ending in –ase Each enzyme has classification number consisting of four digits: EC: 2.3.4.2 First digit refers to a class of enzyme, second - to a subclass, third – to a subsubclass, and fourth means the ordinal number of enzyme in subsubclass

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

24. 2. Transferases Catalyze group transfer reactions

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

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

27. 5. Isomerases Catalyze isomerization reactions

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

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

30. - At a fixed enzyme concentration [E], the initial velocity Vo is almost linearly proportional to substrate concentration [S] when [S] is small but is nearly independent of [S] when [S] is large - Rate rises linearly as [S] increases and then levels off at high [S] (saturated) Rate of Catalysis

31. The basic equation derived by Michaelis and Menten to explain enzyme-catalyzed reactions is V max [S] v o = K m + [S] The Michaelis-Menten Equation K m - Michaelis constant; V o – initial velocity caused by substrate concentration, [S]; V max – maximum velocity

32. Effect of enzyme concentration [E] on velocity (v) In fixed, saturating [S], the higher the concentration of enzyme, the greater the initial reaction rate This relationship will hold as long as there is enough substrate present

33. 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

34. 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 Reversible inhibition

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

36. 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 Noncompetitive inhibition

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

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

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

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

41. 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 Suicide inhibitors

42. 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 Allosteric enzymes

43. 2 types of modulators (inhibitors or activators) Negative modulator (inhibitor) –binds to the allosteric site and inhibits the action of the enzyme –usually it is the end product of a biosynthetic pathway - end-product (feedback) inhibition Positive modulator (activator) –binds to the allosteric site and stimulates activity –usually it is the substrate of the reaction

44. 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 Example of allosteric enzyme - phosphofructokinase-1 (PFK-1) PEP

45. 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

46. 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 Isoenzymes (isozymes) Some metabolic processes are regulated by enzymes that exist in different molecular forms - isoenzymes

47. H 4 : highest affinity; best in aerobic environment M 4 : lowest affinity; best in anaerobic environment Isoenzymes are important for diagnosis of different diseases There are 5 Isozymes of LDH:  H 4 – heart  HM 3  H 2 M 2  H 3 M  M 4 – liver, muscle Lactate dehydrogenase – tetramer (four subunits) composed of two types of polypeptide chains, M and H Example: lactate dehydrogenase (LDH) Lactate + NAD + pyruvate + NADH + H +

48. Product of a pathway controls the rate of its own synthesis by inhibiting an early step (usually the first “committed” step (unique to the pathway) Feedback inhibition Metabolite early in the pathway activates an enzyme further down the pathway Feed-forward activation

49. Stages of metabolism Catabolism Stage I. Breakdown of macromolecules (proteins, carbohydrates and lipids to respective building blocks. Stage II. Amino acids, fatty acids and glucose are oxidized to common metabolite (acetyl CoA) Stage III. Acetyl CoA is oxidized in citric acid cycle to CO 2 and water. As result reduced cofactor, NADH 2 and FADH 2, are formed which give up their electrons. Electrons are transported via the tissue respiration chain and released energy is coupled directly to ATP synthesis.

50. Glycerol Catabolism

51. Catabolism is characterized by convergence of three major routs toward a final common pathway. Different proteins, fats and carbohydrates enter the same pathway – tricarboxylic acid cycle. Anabolism can also be divided into stages, however the anabolic pathways are characterized by divergence. Monosaccharide synthesis begin with CO 2, oxaloacetate, pyruvate or lactate. Amino acids are synthesized from acetyl CoA, pyruvate or keto acids of Krebs cycle. Fatty acids are constructed from acetyl CoA. On the next stage monosaccharides, amino acids and fatty acids are used for the synthesis of polysaccharides, proteins and fats.

52. Compartmentation of metabolic processes permits: - separate pools of metabolites within a cell - simultaneous operation of opposing metabolic paths - high local concentrations of metabolites Example: fatty acid synthesis enzymes (cytosol), fatty acid breakdown enzymes (mitochondria) Compartmentation of Metabolic Processes in Cell

53. Compartmentation of metabolic processes

54. Pyruvate formed in the aerobic conditions undergoes conversion to acetyl CoA by pyruvate dehydrogenase complex. Pyruvate dehydrogenase complex is a bridge between glycolysis and aerobic metabolism – citric acid cycle. Pyruvate dehydrogenase complex and enzymes of cytric acid cycle are located in the matrix of mitochondria. OXIDATIVE DECARBOXYLATION OF PYRUVATE

55. Pyruvate translocase, protein embedded into the inner membrane, transports pyruvate from the intermembrane space into the matrix in symport with H + and exchange (antiport) for OH -. Entry of Pyruvate into the Mitochondrion Pyruvate freely diffuses through the outer membrane of mitochon- dria through the channels formed by transmembrane proteins porins.

56. Pyruvate dehydrogenase complex (PDH complex) is a multienzyme complex containing 3 enzymes, 5 coenzymes and other proteins. Conversion of Pyruvate to Acetyl CoA Pyruvate dehydrogenase complex is giant, with molecular mass ranging from 4 to 10 million daltons. Electron micrograph of the pyruvate dehydrogenase complex from E. coli.

57. Enzymes: E1 = pyruvate dehydrogenase E2 = dihydrolipoyl acetyltransferase E3 = dihydrolipoyl dehydrogenase Coenzymes: TPP (thiamine pyrophosphate), lipoamide, HS-CoA, FAD+, NAD+. TPP is a prosthetic group of E1; lipoamide is a prosthetic group of E2; and FAD is a prosthetic group of E3. The building block of TPP is vitamin B 1 (thiamin); NAD – vitamin B 5 (nicotinamide); FAD – vitamin B 2 (riboflavin), HS-CoA – vitamin B 3 (pantothenic acid), lipoamide – lipoic acid

58. Overall reaction of pyruvate dehydrogenase complex Pyruvate dehydrogenase complex is a classic example of multienzyme complex The oxidative decarboxylation of pyruvate catalized by pyruvate dehydrogenase complex occurs in five steps.

59. Glucose Glucose-6- phosphate Pyruvate Glycogen Ribose, NADPH Pentose phosphate pathway Synthesis of glycogen Degradation of glycogen Glycolysis Gluconeogenesis LactateEthanol Acetyl Co A Fatty Acids Amino Acids The citric acid cycle is the final common pathway for the oxidation of fuel molecules — amino acids, fatty acids, and carbohydrates. Most fuel molecules enter the cycle as acetyl coenzyme A.

60. Names: The Citric Acid Cycle Tricarboxylic Acid Cycle Krebs Cycle In eukaryotes the reactions of the citric acid cycle take place inside mitochondria Physiology or Medicine. Hans Adolf Krebs. Biochemist; born in Germany. Worked in Britain. His discovery in 1937 of the ‘Krebs cycle’ of chemical reactions was critical to the understanding of cell metabolism and earned him the 1953 Nobel Prize for Physiology or Medicine.

61. An Overview of the Citric Acid Cycle A four-carbon oxaloacetate condenses with a two-carbon acetyl unit to yield a six-carbon citrate. An isomer of citrate is oxidatively decarboxylated and five-carbon  - ketoglutarate is formed.  -ketoglutarate is oxidatively decarboxylated to yield a four-carbon succinate. Oxaloacetate is then regenerated from succinate. Two carbon atoms (acetyl CoA) enter the cycle and two carbon atoms leave the cycle in the form of two molecules of carbon dioxide. Three hydride ions (six electrons) are transferred to three molecules of NAD +, one pair of hydrogen atoms (two electrons) is transferred to one molecule of FAD. The function of the citric acid cycle is the harvesting of high- energy electrons from acetyl CoA.

62. 1. Citrate Synthase Citrate formed from acetyl CoA and oxaloacetate Only cycle reaction with C-C bond formation Addition of C 2 unit (acetyl) to the keto double bond of C 4 acid, oxaloacetate, to produce C 6 compound, citrate citrate synthase

63. 2. Aconitase Elimination of H 2 O from citrate to form C=C bond of cis-aconitate Stereospecific addition of H 2 O to cis-aconitate to form isocitrate aconitase

64. 3. Isocitrate Dehydrogenase Oxidative decarboxylation of isocitrate to a-ketoglutarate (a metabolically irreversible reaction) One of four oxidation-reduction reactions of the cycle Hydride ion from the C-2 of isocitrate is transferred to NAD + to form NADH Oxalosuccinate is decarboxylated to a-ketoglutarate isocitrate dehydrogenase

65. 4. The  -Ketoglutarate Dehydrogenase Complex Similar to pyruvate dehydrogenase complex Same coenzymes, identical mechanisms E 1 - a-ketoglutarate dehydrogenase (with TPP) E 2 – dihydrolipoyl succinyltransferase (with flexible lipoamide prosthetic group) E 3 - dihydrolipoyl dehydrogenase (with FAD)  -ketoglutarate dehydrogenase

66. 5. Succinyl-CoA Synthetase Free energy in thioester bond of succinyl CoA is conserved as GTP or ATP in higher animals (or ATP in plants, some bacteria) Substrate level phosphorylation reaction HS- + GTP + ADP GDP + ATP Succinyl-CoA Synthetase

67. Complex of several polypeptides, an FAD prosthetic group and iron-sulfur clusters Embedded in the inner mitochondrial membrane Electrons are transferred from succinate to FAD and then to ubiquinone (Q) in electron transport chain Dehydrogenation is stereospecific; only the trans isomer is formed 6. The Succinate Dehydrogenase Complex Succinate Dehydrogenase

68. 7. Fumarase Stereospecific trans addition of water to the double bond of fumarate to form L-malate Only the L isomer of malate is formed Fumarase

69. 8. Malate Dehydrogenase Malate Dehydrogenase Malate is oxidized to form oxaloacetate.