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  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.


  3. Enzymes - catalysts of biological reactions Accelerate reactions by a millions fold

  4. Common features for enzymes and inorganic catalysts: 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

  5. 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

  6. 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

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

  8. 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

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

  10. 5,6,7,8 - Tetrahydrobiopterin Cofactor of nitric oxide synthase

  11. 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

  12. 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

  13. 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)

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

  15. 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)

  16. 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)

  17. 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

  18. 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

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

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

  21. 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)

  22. 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-

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

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

  25. 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

  26. 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 hasclassification number consisting of four digits: EC: 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

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

  28. 2.Transferases • Catalyze group transfer reactions

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

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

  31. 5. Isomerases • Catalyze isomerization reactions

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

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

  34. Rate of Catalysis • 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)

  35. Leonor Michaelis and Maud Menten – first researchers who explained the shape of the rate curve (1913) During reaction enzyme molecules, E, and substrate molecules, S, combine in a reversible step to form an intermediate enzyme-substrate (ES) complex k1 k2 E + S ES E + P k-1 k-2 k1, k-1, k2, k-2 - rate constant- indicate the speed or efficiency of a reaction

  36. Vmax[S] vo = Km + [S] The Michaelis-Menten Equation The basic equation derived by Michaelis and Menten to explain enzyme-catalyzed reactions is Km- Michaelis constant; Vo – initial velocity caused by substrate concentration, [S]; Vmax – maximum velocity

  37. 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

  38. 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

  39. 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

  40. 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

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

  42. 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

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

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

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

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

  47. 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

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

  49. 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 modulatorsbind noncovalently to allosteric site and regulate enzyme activity via conformational changes

  50. 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