ENZYME- BIOLOGICAL CATALYST

1. ENZYME-BIOLOGICAL CATALYST

2. Enzyme As Catalyst • All enzymes are proteins - with the exception of some RNAs that catalyze their own splicing all enzymes are proteins • In general, names end with suffix “ase” - tyrosinase (tyrosine), celullase (cellulose), protease (protein), lipase (lipid) • Enzyme: a biological catalyst • enzymes can increase the rate of a reaction by a factor of up to 1020 over an uncatalyzed reaction

3. Catalysis: - the process of increasing the rate of chemical reactions • Catalyst: - the substance that facilitate in catalysis

4. Enzyme As Catalyst • Enzymes are catalysts: - increase the rate of a reaction - not consumed by the reaction - act repeatedly to increase the rate of reaction - enzymes often “specific” – promote only 1 particular reaction, others catalyze a family of similar reactions • cellulase – cellulose as substrate • hexokinase – any 6 ring monosaccharide - fructose, glucose

5. General Properties of Enzyme • Higher reaction rates: - the rates of enzymatically catalyzed reactions are 106 to 1020> than uncatalyzed reaction • Milder reaction rates: - enzyme catalyzed reactions occur under relatively milder conditions: < 100oC, atmospheric pressure and nearly neutral pH- contrast with chemical catalysis requires high temperature, pressures and extremes pH. • Greater reaction specificity: - enzyme have greater degree of specificity to their substrates and their products – rarely have side products

6. Enzyme Catalysis Active site - part of enzyme to which the substrate binds and the reaction takes place Substrate – a reactant in an enzyme-catalyzed reaction Product Enzyme-substrate (ES) complex – the intermediate formed when the substrate is bind at the active site of an enzyme

7. Enzyme Catalysis GENERAL FORMULA E = enzyme S = substrate P = product

8. Enzyme catalysis reaction • Physically interact with their substrates to effect catalysis • E + S ES ES* EP E + P • Where: - E = enzyme - ES = enzyme/substrate complex - ES* = enzyme/transition state complex - EP = enzyme/product complex - P = product

9. Enzyme catalysis reaction • Substrate bind to the enzyme’s active site pocket in the enzyme • Catalytic site = active site = where reaction takes place

10. Enzyme catalysis reaction • E + S ES ES* EP E + P • 1st step: enzyme binds to substrate molecule to form an enzyme – substrate complex • E + S ES Enzyme

11. Enzyme catalysis reaction • E + S ES ES* EP E + P • 2nd step: Formation of the transition state complex where the bound substance is neither product nor reactant • ES ES*, ES≠ES

12. Enzyme catalysis reaction • E + S ES ES* EP E + P • 3rd step: Formation of the enzyme – product complex ES* EP

13. Enzyme catalysis reaction • E + S ES ES* EP E + P • 4th step: Release of product EP E + P

14. Enzyme catalysis reaction • Enzyme can only work on one substrate molecule at a time • Not change during the reaction • One product is release, enzyme is available to accept another substrate molecule

15. Enzyme Catalysis • Rate of reaction = reaction velocity (V) - the rate of enzyme reaction is measured by the rate of the appearance of products or the rate of disappearance of substrates. - d[P]/dT or d[S]/dT mol product/min or mol substrate/min • Enzyme activity? • 1 unit (U) is the amount of enzyme that catalyses the reaction of 1 mol of substrate per minute under specified conditions.

16. Enzyme Catalysis • The rate of a reaction depends on its activation energy, DG°‡ • an enzyme provides an alternative pathway with a lower activation energy • Activation energy – the energy required to start a reaction • Transition state – the intermediate stage in a reaction in which the old bonds break and new bonds are formed

17. How enzyme work? • Transition state theory: • The enzyme (E) must approach the substrate (S), the substrate attach to the active site through noncovalent bond • Formed the high energy (unstable) ES complex • In ES complex, the covalent bond in substrate is in the process of breaking while the EP complex is forming.

18. Enzyme Catalysis - Example • Consider the reaction catalase • No catalyst, • with added Fe3+ salt, • with added catalase

19. (a) – a/e for the reaction in the absence of a catalyst • (b) – a/e lowered in the presence of an iron catalyst • (c) – energy diagram for the catalase-catalyzed breakdown of H2O2 • (d) – energy diagram for the noncatalysed breakdown of H2O2 at elevated temperature a/e – activation energy

20. Active site • Has specificity – can discriminate among possible substrate molecules - others recognize a functional group (group specificity) - only recognize one type of molecule (eg. D vs L isomer) (absolute specificity) • Relatively small 3D region within the enzyme - small cleft or crevice on a large protein • Substrates bind in active site by weak non-covalent interactions (Hydrogen bond, hydrophobic and ionic interaction)

21. Active site • The interactions hold the substrate in the proper orientation for most effective catalysis • The energy derived from these interactions – binding energy • Binding energy is used, in large part to lower the activation energy and stabilize the transition state complex (ES*) • Each non-covalent interaction provides energy to stabilize the transition state

22. Binding Models • Two models have been developed to describe formation of the enzyme-substrate complex • Lock-and-key model: substrate binds to that portion of the enzyme with a complementary shape • Induced fit model: binding of the substrate induces a change in the conformation of the enzyme that results in a complementary fit

23. Enzyme/substrate interaction • Lock and key model - substrate (key) fits into a perfectly shaped space in the enzyme (lock) - lots of similarities between the shape of the enzyme and the shape of the substrate - highly stereospecific - implies a very RIGID inflexible active site - site is preformed and RIGID

24. Enzyme/substrate interaction • Induced fit model (hand in glove analogy) - count the flexibility of proteins - substrate fits into a general shape in the enzyme, causing the enzyme to change shape (conformation); close but not perfect fit of E + S - change in protein configuration leads to a near perfect fit of substrate with enzyme Figure 6.3, pg 148 Campbell & Farrell, Biochemistry, 6th Ed., 2009, Thomson Brooks/Cole

25. Key words for today • You need to understand: • Factors effecting enzyme reaction rate • 6 classes of enzymes • Cofactor, coenzymes, holoenzymes, apoenzymes • Allosteric enzyme, effectors (positive and negative), heterotropic and homotropic allosterism • Isoenzyme and multienzyme

26. Characteristics of enzyme reactions • What influence the enzyme reaction rate? • Substrate concentration • Temperature • pH • Enzyme concentration • Inhibitor

27. Characteristics of enzyme reactions • Substrate Saturation: Increasing the [substrate] increases the rate of reaction (enzyme activity). • enzyme saturation limits reaction rates. An enzyme is saturated when the active sites of all the molecules are occupied most of the time. • At the saturation point, the reaction will not speed up, no matter how much additional substrate is added. The graph of the reaction rate will plateau. [substrate] = substrate concentration

28. Characteristics of enzyme reactions • Temperature - very sensitive to temperature changes - low temp, rate of an enzyme-catalysed reaction increases proportionally with increasing temperature

29. Characteristics of enzyme reactions • Effects of Temperature: • All enzymes work within a range of temperature specific to the organism. • Increases in temperature lead to increases in reaction rates - is a limit to the increase because higher temperatures lead to a sharp decrease in reaction rates - due to the denaturating (alteration) of protein structure resulting from the breakdown of the weak ionic and hydrogen bonding that stabilize the three dimensional structure of the enzyme.

30. Characteristics of enzyme reactions • pH - enzymes have an optimal pH at which they function properly - varies to each other but most in the range of pH 6-8 pepsin in the stomach works best at a pH of 2 and trypsin at a pH of 8.

31. Characteristics of enzyme reactions • Effects of pH: Most enzymes are sensitive to pH and have specific ranges of activity. • All have an optimum pH. The pH can stop enzyme activity by denaturating (altering) the three dimensional shape of the enzyme by breaking ionic, and hydrogen bonds.

32. Characteristics of enzyme reactions • Enzyme concentration - the higher the concentration, the greater should be the initial reaction rate – will be lasting as long as substrate present

33. Characteristics of enzyme reactions • Inhibitor - inhibit enzyme by occupy the active site or bind to other part of enzyme – leading to the change of enzyme shape and eventually the active site - this will decrease the enzymatic reaction rate

34. Classification of Enzymes • Have 6 categories • Each enzyme has an official international name ending with –ase and a classification number • Number consists in 4 digits (referred to a class and subclass of reaction

35. Classification of Enzymes

36. Classification of Enzymes Table 5.1, pg 136 Boyer, R., Concepts in Biochemistry, 3rd Ed., 2006, John Wiley &Sons

37. Enzyme Classes: Examples More examples:Refer Table 5.2, pg 136 , Boyer, R., Concepts in Biochemistry, 3rd Ed., 2006, John Wiley &Sons

38. Enzyme Classes: Examples

39. Enzymes & cofactor • Enzymes require chemical entity in order to function properly (assists an enzyme in catalytic action) • Cofactor – nonprotein molecule that assist in an enzyme catalytic reaction • Coenzyme – smaller organic or organometallic molecule derived from vitamin, weakly bound to enzyme, temporarily associated with enzymes • Prosthetic group – coenzymes that are covalently or noncovalently tightlybound to enzyme and always present.

40. Enzymes & cofactor • Holoenzyme – an enzyme in its complete form including polypeptide(s) and cofactor • Apoenzyme – enzyme in its polypeptide form without any necessary prosthetic groups or cofactors

41. Allosteric Enzymes • Allosteric enzyme: an oligomer whose biological activity is affected by other substances binding to it • these substances change the enzyme’s activity by altering the conformation(s) of its 4°structure • Allosteric effector: a substance that modifies the behavior of an allosteric enzyme; may be an • allosteric inhibitor = negative effectors • allosteric activator = positive effectors

42. Allosteric enzyme in feedback Inhibition Formation of product inhibits its continued production – feedback inhibition

43. Allosteric Enzymes (Cont’d) • The key to allosteric behavior is the existence of multiple forms for the 4°structure of the enzyme • allosteric effector: a substance that modifies the 4° structure of an allosteric enzyme • homotropic effects: allosteric interactions that occur when several identical molecules are bound to the protein; e.g., the binding of aspartate to ATCase • heterotropic effects: allosteric interactions that occur when different substances are bound to the protein; e.g., inhibition of ATCase by CTP and activation by ATP ATCase = aspartate transcarbomylase CTP = cytidine triphosphate

44. Allosteric enzymes • A change in conformational structure at one location of a multisubunit protein that causes a conformational change at another location on the protein • Effectors – i) serves as stimulants to enzyme (+ve effectors) = increase catalytic activity– ii) inhibitors (-ve effectors) to enzyme = reduce/inhibit catalytic activity - Act by reversible, noncovalent binding to a site on the enzyme • Larger and more complex than nonallosteric enzyme • Have 2 or more subunits (oligomeric) • Allosteric enzymes have regulatory sites for binding of substrates and reaction (catalytic sites)

45. HOMOTROPIC ALLOSTERISM • Eg. Tetrameric allosteric enzyme composed of 4 identical subunits • Each subunit has a catalytic site where substrate/effector will bound and transformed to product • Once bound to active site, a message will transmitted via conformational changes to an active site on another subunit which makes it easier for a substrate molecule to bind and react at that site • This type (substrate and effector) are the same is called cooperative or homotropic

46. HETEROTROPIC ALLOSTERISM • A dimer with nonidentical subunits • Subunit α contain the active site – catalytic subunit • Subunit β contains the site for effector binding – regulatory subunit • Binding of a specific effector molecule to the regulatory site on the β subunit sends a signal via conformational changes to the catalytic site on subunit α • Substrate and effector different kinds of molecules - heterotropic

47. Isoenzymes • Enzymes that catalyze the same reaction (catalytically and structurally similar) but are encoded by different genes • Glycogen phosphorylase-synthesize in liver, brain and muscle-involves in degradation of glycogen • Isoenzymes = isoforms

48. Multienzymes • A group of noncovalently associated enzymes that catalyze 2 or more sequential steps in metabolic/biochemical pathway