Enzymes bph

Enzymes bph

1. ENZYMES MAN BAHADUR RANA BPH ACAS,NEPAL

2. Enzymes: Introduction Enzymes may be defined as biocatalysts synthesized by living cells. They are protein in nature (exception - RNA acting as ribozyme ), colloidal and thermolabile in character, and specific in their action. Enzyme or catalysts accelerates reaction by lowering energy of activation of the reacting molecules but do not influence their thermodynamic characteristics. Similarly catalyst or enzymes do not change the equilibrium constants of reactions.

3. Catalysts or enzymes increase product formation by (1) lowering the energy barrier (activation energy) for the product to form (2) increases the favorable orientation of colliding reactant molecules for product formation to be successful (stabilize transition state intermediate)

4. Structural Organization of Enzyme Some enzymes require an additional chemical component called a cofactor for their activity holoenzyme = Apoenzyme or Apoprotein + Co-factors (Active) (Inactive) Haloenzyme : A complete catalytically active enzyme with its cofactor is called Holoenzyme . Apoenzyme : The protein part of the holoenzyme is called apoenzyme .

5. Structural Organization of Enzyme Cofactors are non-protein molecules that help enzymes function and associated with the active site of enzyme. Organic co-factors – thiamin, riboflavin, niacin, biotin are called Co-enzymes. Coenzymes are often regarded as the second substrates or co-substrates. Inorganic co-factors – Mg ++ , Fe ++ , Zn ++ , Mn ++ are called activators. In some enzymes, Co-enzyme or metal ion is tightly and permanently bound to the enzyme called a prosthetic group.

6. Structural Organization of Enzyme Active Centre/Active Site As the substrate molecules are comparatively much smaller than the enzyme molecules, there should be some specific regions or sites on the enzyme for binding with the substrate. Such sites of attachment are variously called as ‘active sites’ or ‘catalytic sites’ or ‘substrate sites’.

7. Structural Organization of Enzyme Isozyme : Many enzymes occur in more than one molecular form in the same species, in the same tissue or even in the same cell and catalyze the same reaction at different rates. Their activities towards different substrates will also vary . Lactate dehydrogenase -have 4 subunits hence 5 isozymes LDH1 and LDH2 have greater activity towards Beta- Hydroxy butyrate than others.

8. Classification of Enzymes Since 1964, the IUB system of enzyme classification has been in force. Enzymes are divided into six major classes (in that order). Each class on its own represens the general Type of reaction brought about by the enzymes of that class.

9. Classes of enzymes Oxidoreductases = catalyze oxidation-reduction reactions e.g. Succinate dehydrogenase , Alcohol dehydrogenase , dehydrogenase of NADH and NADPH Transferases = catalyze transfer of functional groups from one molecule to another. eg . Phosphotransferase , aminotransferase , Acyl transferase Hydrolases = catalyze hydrolytic cleavage. eg . Peptidase, Glycosidase, Esterase.

10. Classes of enzymes 4. Lyases = catalyze removal of a group from or addition of a group to a double bond, or other cleavages involving electron rearrangement. eg . Decarboxylases , Aldolase , Dehydratase , Deaminase . 5. Isomerases = catalyze intramolecular rearrangement. Eg . Glucose-6- phosphoisomerase , Phosphoglycerate phosphomutase . 6. Ligases = catalyze reactions in which two molecules are joined. Eg . Amino acetyl tRNA synthetase , Glutamine synthetase .

11. Mechanism of Enzymes Action For any chemical reaction to occur, the reactants have to be in an activated state or transition state which depends upon mainly two factors. 1. Lowering of Activation energy : The energy required by the reactants to undergo the reaction is known as activation energy. The reactants when heated attain the activation energy. The catalyst (or the enzyme in the biological system) reduces the activation energy and this causes the reaction to proceed at a lower temperature.

12. Mechanism of Enzymes Action Enzymes do not alter the equilibrium constants, they only enhance the velocity of the reaction. The role of catalyst or enzyme is comparable with a tunnel made in a mountain to reduce the .barrier

13. Mechanism of Enzymes Action Enzyme Substrate Complex Formation Michaelis Menten have proposed a hypothesis for enzyme action, which is most acceptable. According to their hypothesis, the enzyme molecule (E) first combines with a substrate molecule (S) to form an enzyme-substrate ES complex which further dissociates to form product (P) and enzyme (E) back. Enzyme once dissociated from the complex is free to combine with another molecule of substrate and form product in a similar way. The ES complex is an intermediate or transient complex and the bonds involved are weak non-covalent bonds such as H-bond, Van der waals forces, hydrophobic interations . Sometimes two substrates can bind to an enzyme molecule and such reactions are called as bisubstrate reactions.

14. Mechanism of Enzymes Action A few theories have been put forth to explain mechanism of enzyme-substrate complex formation . Template or Lock and key model or Fischer's template theory: It states that the active site already exists in proper conformation even in absence of substrate. Thus the active site by itself provides a rigid, pre-shaped template fitting with the size and shape of the substrate molecule. Substrate fits into active site of an enzyme as the key fits into the lock and hence it is called lock-and-key model. But this can not explain change in enzymatic activity in presence of allosteric modulators.

15. Mechanism of Enzymes Action

16. Mechanism of Enzymes Action 2. Induced fit theory or Koshland's model Koshland, in 1958, proposed a more acceptable and realistic model for enzyme substrate complex formation. The important feature of this model is the flexibility of the region of active site. According to this active site does not possess a rigid preformed structure on enzyme to fit the substrate. On the contrary, the substrate during its binding induces conformational changes in the active site to attain the final catalytic shape and form. This explains several matters related to enzyme actions such as enzymes become inactive on denaturation,, allosteric modulation and competitive inhibition.

17. Mechanism of Enzymes Action 3. Substrate strain theory In this model, the substrate is strained due to the induced conformation change in the enzyme. It is also possible that when a substrate binds to the preformed active site, the enzyme induces a strain to the substrate. The strained substrate leads to the formation of product. In fact, a combination of the induced fit model with the substrates train is considered to be operative in the enzymatic action.

18. Factors Affecting Enzyme Action a.Temperature :A bell-shaped curve is usually observed. Temperature coefficient or Q 10 is defined as increase in enzyme velocity when the temperature is increased by 10 o C. 1.Increase of velocity with temperature : The reaction velocity increases with temperature until a peak velocity is reached. This increase is the result of the increased number of molecules having sufficient energy to pass over the energy barrier and form the products of the reaction.

19. Factors Affecting Enzyme Action 2. Decrease of velocity with higher temperature: Further elevation of the temperature results in a decrease in reaction velocity as a result of temperature-induced denaturation of the enzyme

20. Factors Affecting Enzyme Action b.pH Increase in the hydrogen ionconcentration (pH) considerably influences the enzyme activity and a bell-shaped curve is normally obtained

21. Factors Affecting Enzyme Action Effect of pH on the ionization of the active site: The concentration of H+affects reaction velocity in several ways. First, the catalytic process usually requires that the enzyme and substrate have specific chemical groups in either an ionized or unionized state in order to interact. For example, catalytic activity may require that an amino group of the enzyme be in the protonated form –NH 3 + At alkaline pH this group is deprotonated , and the rate of the reaction, therefore, declines. 2. Effect of pH on enzyme denaturation: Extremes of can also lead to denaturation of the enzyme, because the structure of the catalytically active protein molecule depends on the ionic character of the amino acid side chains

22. Factors Affecting Enzyme Action 3.The pH optimum varies for different enzymes: The pH at which maximal enzyme activity is achieved is different for different enzymes, and often reflects the [H+] at which the enzyme functions in the body. For example, pepsin, a digestive enzyme in the stomach, is maximally active at pH 2, whereas other enzymes, designed to work at neutral pH, are denatured by such an acidic environment .

23. Factors Affecting Enzyme Action c. Effect of Enzyme Concentration As the concentration of the enzyme is increased, the velocity of the reaction proportionately increase as enzyme is the limiting factor in the enzyme substrate Reaction. This property of enzyme is made use in determining the serum enzymes for the diagnosis of diseases. By using a known volume of serum, and keeping all the other factors (substrate, pH, temperature etc.) at the optimum level, the enzyme could be assayed in the laboratory.

24. Factors Affecting Enzyme Action Effect of Substrate Concentration: Increase in the substrate concentration gradually increases the velocity of enzyme reaction within the limited range of Substrate levels. A rectangular hyperbola is obtained when velocity is plotted against the substrate concentration, Three distinct phases of the reaction are observed in the graph (A- linear; B-curve; C-almost unchanged). Order of reaction : When the velocity of the reaction is almost proportional to the substrate concentration( i.e. [S] is less than Km),the rate of the reaction is said to be first order with respect to substrate. When the [S] is much greater than Km, the rate of reaction is independent of substrate concentration, and the reaction is said to be zero order.

25. Factors Affecting Enzyme Action

26. Factors Affecting Enzyme Action e. Effect of Product Concentration: The accumulation of reaction products generally decreases the enzyme velocity. For certain enzymes, the products combine with the active site of enzyme and form a loose complex and, thus, inhibit the enzyme activity. It is also possible that under certain conditions of high concentration of products a reverse reaction may be favored forming back the substrate. In the living system, this type of inhibition is generally prevented by a quick removal of products formed.

27. Factors Affecting Enzyme Action f. Effect of time: Under ideal and optimal conditions (like pH, temperature etc.), the time required for an enzyme reaction is less. Variations in the time of the reaction are generally related to the alterations in pH and temperature. g. Other factors : Effect of Activators and Coenzymes, Effect of Modulators and Inhibitors, Effect of light and radiation.