1.
BIOCHEMICAL PRINCIPLES OFENZYME ACTION Presented by Dr. B. Victor., Ph.D. Email: bonfiliusvictor @gmail. com Blog: bonvictor.blogspot.com

2.
Presentation outline Definition and explanation Properties, origin, number/cell and size of enzymes. All enzymes are proteins-evidences Enzyme catalysts and chemical catalysts. Classification and naming of enzymes. Principle-active site-characteristics. Mechanism of enzyme catalysis-models. Activation energy and equilibrium constant. Enzyme kinetics-Michaelis constant Properties of enzyme. Coenzymes, isoenzymes, proenzymes and allosteric enzymes

3.
Definition and explanation Enzymology is the study of enzymes • Study of understanding the nature and functions of enzymes. Catalysts • Catalysts are chemicals that speed up the rate of biochemical reactions.

4.
Definition of enzymes Enzymes • Enzymes are proteins functioning as catalysts that speed up reactions by lowering the activation energy. • The enzyme catalysts regulate the structure and function of cells and organisms.

5.
Properties of enzymes Enzymes catalyze Enzymes accelerate Enzymes decrease the biochemical the velocity of a energy of activationreactions in living cell. biochemical reaction of substrates. The chemical nature of Enzyme does not an enzyme is not change the equilibrium changed by entering constant of a reaction a biochemical reaction

6.
Origin of enzyme chemistry In 1850s, Louis Pasteur presented a theory that sugar is converted into ethanol in yeast by ‘ferments’. He recognized that ferments was acting as a catalyst in fermentation The word ‘enzyme’(en=in; zyme-= yeast) was coined by German physiologist Wilhelm Kuhne in 1877. Enzyme literally means ‘in yeast’. In 1897 Eduard Buchner discovered that fermentation of the sugar was possible with the enzyme ’zymase’. He received the Nobel prize for chemistry in 1907.

7.
Number of enzymes per cell A bacterium like Escherichia coli has about 4228 proteins of which almost 1701 of them are enzymes. Mammals have more than 10 times the number of proteins and enzymes found in E. coli. The human body makes approx. 22 digestive enzymes .

8.
Size of enzymes Enzymes are globular proteins with three dimensional shape. Size range from 62 amino acid residues to over 2,500 in animal fatty acid synthetase. Molecular mass 12 kd to 1000 kd.

9.
“All enzymes are proteins”- justification UV absorption at 280nm Have peptide Amphoteric bonds nature Influenced by pH Have isoelectric and temperature point nondialysable

10.
Difference between enzyme catalysts and chemical catalysts enzyme catalysts chemical catalysts• Protein in nature • Non-protein in nature• Catalyses a specific reaction • Catalyze different reactions• Catalysis occur via active site • Catalysis takes part as a of enzymes. whole.• The enzyme does not return • Catalyst always return to its to their original state after a biochemical reaction. original state.• Generally produced by living • Reacts outside living cells. cells and acts inside living cells.

11.
Classification of enzymesSite action- Source of enzyme- Chemical composition –Intracellular, Plants, animals, Simple, metallo- orextracellular microorganisms conjugated Type of reaction – Type of substrate –protein- protease Hydrolase, Carbohydrate – Oxidase, carbohydrases dehydrogenase Lipids - lipase

12.
6-major classes of enzymesOxidoreductases • Catalyze redox reactions(6-sub classes). • Transfer functional groups between donors and Transferases acceptors. Hydrolases • Catalyze hydrolysis of substrates Lyases • Catalyze removal of a group other than Hydrolysis. Isomerases • Catalyze inter-molecular rearrangement Ligases • Catalyze the union of two molecules.

13.
Enzyme nomenclature Naming the enzymes Named byTo add ‘suffix’to Retain old groups thatthe name of the traditional catalyse similar substrate To add suffix- names- chemical Named from the ’lytic’ to denote Urea-urease reactions species of origin Ptyalin splitting. Arginine- Dehydrogenases Papain-papaya Pepsin Proteolytic arginase Oxidases Ficin-ficus Renin lipolytic Tyrosine- Proteinases tyrosinase trypsin lipases

14.
Each enzyme is represented by 4numbers as per enzyme commission First number class indicates Second number subclass indicates Third number Sub- indicates subclass Fourth Serial number number in indicates that class e.g. lactate dehydrogenase (1.1.1.27)

15.
Principle of enzyme action Enzyme- Enzyme Substrate substrate (E) (S) Complex (ES)Enzyme-Substrate Enzyme ProductsComplex (E) (P) (ES)

16.
Basic principle of enzyme actionMechanism of enzyme action Details of the mechanism • The metabolite on which the enzyme act is the substrate. • The substrate when activated by an enzyme, combines with the enzyme to form the enzyme- substrate complex. • This in turn dissociates releasing products and leaving the enzyme free.

17.
Active site • The site of attachment ofof enzyme substrate is active site. • This site is variously known as active centre, catalytic site or substrate site. • The active site has two components, the catalytic and specificity site. • The active site has two functions-to bind to a specific substrate and to catalyze its the chemical change.

18.
Active and regulatory sites of anenzyme e.g. ribonucleotide reductase

19.
Characteristics of • The active site occupies aactive site small portion of enzyme molecule. • The active site is well defined with 3-D shape and changes its configuration to bind to a substrate. • The active site is made up a few amino acid side chains. • The active site binds to a substrate by weak forces.

20.
Mechanism of • Emil Fisher proposed lock-enzyme catalysis:Lock and Key Model key model in 1898. • The enzyme-substrate union depends on a reciprocal fit between enzyme and substrate. • To stay fit, the active site must have a complementary shape. • This matching resembles the fitting of a key to a lock.

21.
Mechanism of • Induced fit model wasenzyme catalysis: proposed by Koshland in 1958.Induced Fit Model • The substrate – active sites are flexible but structurally not complementary. • The binding of a substrate to the active site is believed to induce a slight alteration in the shape of active site. • After the release of products the active site returns to its original configuration.

22.
Principle of activation energy • The energy of activation in a chemical reaction is a measure of the energy needed for the conversion of the substrate molecules to the reactive state. • The enzyme catalysts are more efficient in lowering the energy of activation. • For e.g. the decomposition of H2O2 require 18kcal/mole of energy without catalyst, but only 2 kcal/mole in the presence of enzyme catalase.

23.
Equilibrium constant of biochemical reaction • Enzymes do not change the equilibrium constant of a reaction , but only decrease the time it takes to reach the equilibrium.

24.
Enzyme Kinetics (The Michaelis-Menten Model)Enzyme Kinetics is the study of the rate of enzyme catalyzed chemical reactions .

25.
Kinetic parameter, km (The Michaelis-Menten Model)• Km is the measure of the affinity of an enzyme for its substrate. It is generally called Michaelis constant.• Km values of enzymes range from 10-1 to 16-6 M.• Km is a constant for a particular set of enzymes and substrate at optimum temperature and pH.• Km is independent of the enzyme concentration.• A high km means weak binding between the enzyme and its substrate and a low km means strong binding.

26.
Kinetic parameter : Vmax• Vmax shows how well the enzyme catalyzes a reaction when the concentration of substrate is high enough so that all enzyme molecules exist as ES complexes.• The maximum rate (Vmax) represents the turnover number of an enzyme.• This rate constant is also called the molecular activity of an enzyme.

27.
Specificity of enzyme action Narrow • Maltase hydrolyzes only maltose Enzyme • Urease acts on urea specificity Broad • Proteases hydrolyze peptide linkages Enzyme • Exopeptidases hydrolyze terminal of protein chain. • Endopeptidases hydrolyze within protein chain specificity • L-amino acid oxidase acts on L-amino acids.stereo specificity • D-amino acid oxidase acts on D-amino acids

28.
Properties of enzymes• Enzymes are dynamic proteins.• Enzyme activity is specific which may be relative or absolute.• Most enzymes are soluble in water.• Enzymes are colloidal in nature.• Small quantity is required for enzyme action• Temperature –dependent activity – Vant Hoff’s law states that the velocity of chemical reaction is at least doubled by a rise of 100C (Q10).• pH dependent activity – enzymatic action is greatly influenced by changes in hydrogen ion concentration.

29.
Effect of temperature on enzyme activity • Temperature greatly affect the activity of enzymes. • If the temperature is increased by 100C , the rate of enzyme action is doubled. • Denaturation of enzymes occur between 40-600C. • Maximum enzyme activity occurs at optimum temperature.

30.
Effect of pH on enzyme activity • Hydrogen ion concentration also have an influence on enzyme activity. • For most enzymes, the effective pH range is 4.0- 9.0. • Beyond these limits, denaturation of enzymes take place. • Optimum pH for pepsin is 2.0 and for trypsin 8.0

31.
Apo Holo coenzymeenzyme enzyme

32.
Prosthetic groups of apoenzyme • Loosely bound metal ions • Zn2+ =carbonic anhydrasecofactors • Cu 2+ = cytochrome oxidase • Strongly bound organic moleculesProsthetic • Heme, flavins, biotin groups • Strongly bound low molecular weight non-protein.coenzymes • TPP, FMN, FAD, NAD, NADP

33.
Isoenzymes• Certain enzymes occur in mult-imolecular forms in the same organism.• They are structurally related but catalyze the same reactions using different kinetic parameters.• E.g. Lactate dehydrogenase (LDH) Alkaline phosphatase Glutamate oxaloacetate trans aminase Creatine phosphokinase

34.
Zymogens or pro-enzymes:-enzymes synthesized in a catalytically inactive form.Zymogens Active enzymes• Prothrombin • Thrombin• Chymotrypsinogen • Chymotrypsin• Trypsinogen • trypsin• Pepsinogen • Pepsin• Procarboxypeptidase • Carboxypeptidase• Prophospholipase • Phospholipase• Proelastase • Elastase• Fibrinogen • Fibrin

35.
Allosteric enzymes• Allosteric enzymes possess one or more allosteric sites (regulatory sites), which are distinct from the catalytic sites.• Allosteric sites specifically binds to the inhibitory modulators.• Inhibitory modulators may be positive or negative• They modulate an enzyme at any substrate concentration.• The degree of inhibition is related to the concentration of the inhibitor.

36.
Enzyme inhibitors Irreversible Competitive inhibitors inhibitors Enzymeinhibitors reversible Uncompetitive inhibitors inhibitors Noncompetitive inhibitors

37.
Meaning of enzyme inhibitors• Irreversible inhibitors – inhibition of enzyme activity by combining with active site.• Competitive inhibitors – inhibition of enzyme activity by competing with active site.• Uncompetitive inhibitors - inhibition of enzyme activity by combining with allosteric site.• Noncompetitive inhibitors – inhibition by binding with both to the free enzyme and ES at the allosteric site.

38.
Research objectives in the study of enzymes Molecular • Structural aspects :Primary, secondary, tertiary and quaternary structure Protein • PH and temperature stability, isoelectric point • Electrophoretic mobility, molecular mass, spectroscopic properties properties. Catalytic • Specificity, kinetic properties, catalytic mechanism • Regulatory mechanism, thermodynamic constants activitiesPhysiological • Cellular location, metabolic role, metabolic flux. function

39.
Summary• Enzymes are dynamic proteins that accelerate biochemical reactions.• Each enzyme acts on a specific reactant, the substrate.• Enzymes are characterized by greater activity, specificity and susceptibility to the influence of pH, temperature and other environmental changes.• Enzymes act in the presence of non-peptide cofactors or coenzymes.• An enzyme lacking its cofactor is called apoenzyme and the active enzyme with its co-factor, the holoenzyme.

40.
 Dr.B.Victor is a highly experienced professor, recently retired from the reputed educational institution- St. Xavier’s College, Palayamkottai, India-627001. He was the dean of sciences, IQAC coordinator and assistant controller of examinations. He has more than 32 years of teaching and research experience He has taught biochemistry at UG and PG levels and guided 12 Ph.D scholars. Send your comments to : bonfiliusvictor@gmail.com