ENZYME PROPERTIES, FUNCTIONS & KINETICS Week 11: 23&24/11/2011

1. ENZYME PROPERTIES, FUNCTIONS & KINETICSWeek 11: 23&24/11/2011 Khadijahhanimabdulrahman School of bioprocess engineering, UniMap Sem 1: 2011/2012

2. LEARNING OUTCOMES DISCUSS properties and classification of enzyme DISCUSS catalytic properties and enzymatic regulation in cellular metabolism.

3. Introduction • Most important functions of proteins- as catalysts • Living processes consist almost entirely of biochemical reactions- without catalysts these reactions would not occur fast enough. • Enzymes have several remarkable properties: • The rates of enzymatically catalyzed reactions are often phenomenally high. • The enzymes are highly specific to the reactions they catalyzed- side products are rarely formed. • Enzymes can be regulated- important consideration in living organisms- must conserve energy and raw materials.

4. How do enzymes work? • Recognize very specific substrates • Perform specific chemical reactions at high speed. - Enzymes make and break specific chemical bonds of the substrates at a faster rate without being consumed in the process. - At the end of each catalytic cycle, the enzyme is free to begin again with a new substrate molecule

5. Active site = a part of an enzyme where substrate bind and undergo a chemical reaction

6. Properties of enzymes Enzymes are generally globular proteins, some are simple proteins and others are conjugated proteins. Catalysts- substance that enhances the rate of a chemical reaction but not permanently altered by the reaction. Catalysts decrease the activation energy required for a chemical reaction – less energy.

7. Transition state occurs at the apex of both reaction pathway Reaction catalyze by enzyme require low activation energy to convert reactant (substrate) from the ground state to the transition state Ground state (stable, low energy form of mol.)

8. Each type of enzyme molecule contain a unique, intricately binding surface called active site Substrate bind to active site which is typically small cleft on a large protein mol. Beside being binding site, amino acid side chains that line the active site actively participate in catalytic process

9. LOCK & KEY Model Introduce by Emil Fischer 1890 illustrate enzyme specificity Each enzyme binds to a single type of substrate (bcoz the active site and the substrate have complementary structure) active site = substrate binding surface The correct substrate fits into the active site of the enzyme like a key into a lock. Only the right key fits.

11. INDUCED-FIT model Modification of lock and key model by Daniel Koshland (1958) Flexible structure of protein is taken into account In this model, substrate does not fit precisely into a rigid active site Noncovalent interaction between enzyme & substrate change the active site structure, make it fit for the substrate

13. CLASSIFICATION OF ENZYMES Enzymes named according to the type of chemical reaction it catalyzes Six major enzyme categories : 1) Oxidoreductase - Catalyze oxidation-reduction (redox) reaction (enzyme that catalyzes the transfer of electron from 1 molecule to the other) - Subclasses – dehydrogenases, oxidases, oxygenases, reductases, peroxidases & hydroxylases

14. 2) Transferases - catalyze reactions that involve the transfer of groups from one molecule to another - eg. Transaminases, transcarboxylases

15. 3) Hydrolases - cleave bonds by adding water - eg. Phosphatases, peptidases, esterase

16. 4) Lyase - catalyze reactions in which groups (eg. H20, CO2, NH3) removed to form a double bond or added to a double bond. - eg. Decarboxylases, hydratases, deaminases

17. 5) Isomerases • catalyze intramolecular rearrangements. The epimerases catalyze the inversion of asymmetric carbon atoms. • Mutases catalyze the intramolecular transfer of functional group. • --- eg. epimerases or mutases

18. 6) Ligases - catalyze bond formation between two substrate molecules - eg. Synthetase, carboxylase

19. ENZYME KINETICS Enzyme kinetics is the quantitative study of enzyme catalysis (chemical reaction catalyzed by enzyme). Kinetic studies measure reaction rates and the affinity of enzymes for substrates and inhibitors Studying an enzyme's kinetics in this way can reveal the catalytic mechanism of this enzyme, its role in metabolism, how its activity is controlled, and how a drug or a poison might inhibit the enzyme.

20. The rate or velocity of a biochemical reaction is defined as the change in the concentration of a reactant or product per unit time. Another useful term to describe reaction- reaction’s order. Order- determined empirically (experimentation). A reaction is said to follow first-order kinetics- rate depends on the concentration of a single reactant.

22. For a given enzyme concentration and for relatively low substrate concentrations, the reaction rate increases linearly with substrate concentration the enzyme molecules are largely free to catalyze the reaction increasing substrate concentration means an increasing rate at which the enzyme and substrate molecules encounter one another. However, at relatively high substrate concentrations, the reaction rate is zero-order with respect to substrate. the enzyme active sites are almost all occupied

23. MICHAELIS-MENTEN KINETICS Models to investigate the enzyme rates Proposed by Leonor Michaelis and Maud Menten in 1913. When the substrate S binds in the active site of an enzyme E, an intermediate complex (ES) is formed. During the transition state, the substrate is converted into product. After sometimes, the product dissociates from the enzyme.

24. Rate equations for an enzyme catalyzed reaction support a theory involving the formation of ES complexes. At high [S], S saturates E, and the reaction rate is independent of the [S]. The value of v under this condition is called the maximum velocity, Vmax. At low [S], the reaction is first-order with respect to S. The plot of v versus [S] from low to high [S] is a rectangular hyperbola. The rate equation (Michaelis-Menten equation) that describes this relationship is

25. Vmax- the maximum velocity that the reaction can attain The concentration of substrate that corresponds to half-maximum velocity is called the Michaelis constant, Km. The enzyme is half-saturated when [S] = Km. Km is a measure of the substrate affinity for enzyme. A small Km indicates high affinity- the rate will approach Vmax more quickly. Km=small value, the enzyme can achieves max. catalytic efficiency at low substrate conc- efficiency of enzyme to convert substrate into product. Michaelis-Menten plot

26. Lineweaver-Burk Plots Km and Vmax values for an enzyme- determined by measuring initial reaction velocities at various substrate conc. Approximate values of Km and Vmax can be obtained by constructing a graph. The Michaelis-Menten equation, hyperbola graph: Can be rearranged by taking it reciprocal:

27. In Lineweaver-Burk plot- generated straight line, y=mx + b ( y and x- 1/v and 1/[S], respectively-variables) m and b are constants ( Km/Vmax and 1/Vmax, respectively) The intercept on the vertical axis is 1/Vmax The intercept on horizontal axis is -1/Km.

28. ENZYME INHIBITION • Enzyme activity- can be inhibited • Molecules that can reduced enzyme activity- inhibitors- drugs, antibiotics, food preservatives and poisons. • Investigation of enzyme inhibitors, important: 1) Enzyme inhibition- important means by which metabolic pathways are regulated. 2) Numerous clinical therapies based on enzyme inhibition. • Eg: antibiotics and drugs reduce /eliminate the activity of specific enzymes. • The most effective AIDS treatment- multidrug therapy that includes protease inhibitors- molecules that disable a viral enzyme required to make new virus.

29. 3) Enzyme inhibition- enable biochemist to develop techniques to probe physical and chemical structure and functional properties of enzymes. • Enzyme inhibition occur when- a compound competes with substrate for the active site of the free enzyme. • 3 classes of enzyme inhibitors- competitive, uncompetitive and noncompetitive inhibitors.

30. COMPETITIVE INHIBITORS Competitive inhibitors bind reversibly to free enzyme, not the ES complex, to form an enzyme-inhibitor (El) complex. Substrate and inhibitor compete for the same site on the enzyme

32. Substance that behave as competitive inhibitors, reduce enzyme affinity for substrate Enzyme activity decline, no reaction occur when EI complex exists Effect of competitive inhibitors on enzyme activity is reversed by increasing [S] At high [S], all active site filled with substrate and reaction velocity reaches the value observed without an inhibitor Substances that behave as competitive inhibitors- reduce the enzyme’s affinity for substrate- similar in structure with substrate. With competitive inhibition- Vmax stays constant, Km increases. Shown in the double-reciprocal plot as shift in horizontal intercept.

33. UNCOMPETITIVE INHIBITORS The inhibitors binds only to the enzyme-substrate complex (ES), and not the free enzyme

35. In uncompetitive inhibition- both Km and Vmax are changed, although their ratio are still the same.

36. NONCOMPETITIVE INHIBITORS In some enzyme-catalyzed reactions, an inhibitors can bind to both enzyme and enzyme-substrate complex (EI)

37. A noncompetitive inhibitor binds at a site other than the active site of the enzyme Inhibitor binding result in a modification of enzyme’s conformation that prevent product formation-reduces the enzyme activity. Noncompetitive inhibitors do not affect substrate binding & have little structural resemblance to substrate Is only partially reversed by increasing the substrate concentration

39. In noncompetitive Vmax decreases and Km stays constant The vertical intercept is shifted.

40. SUMMARY FOR ENZYME INHIBITION

41. CATALYTIC MECHANISMS • Enzyme catalysis is the catalysis of chemical reactions by specialized proteins known as enzymes • Despite extensive research, only a few of enzymes mechanism in known. • Enzymes use the same catalytic mechanisms as nonenzymaticcatalysts. • Enzyme achieve higher catalytic rates because their active site possess structure that uniquely suited to promote catalysis • Factors contribute to enzyme catalysis : • proximity and strain effects • electrostatics effects • acid-base catalysis • covalent catalysis

42. FACTOR INFLUENCING CATALYSIS PROXIMITY AND STRAIN EFFECTS For a biochemical reaction to occur, the substrate must closely approach the catalytic site with proper orientation Once substrate correctly positioned, result in a strained enzyme-substrate complex. This strain help to bring the enzyme-substrate complex into the transition state In general, the more tightly the active site can bind the substrate while it is in the transition state, the greater the reaction rate. When an enzyme and substrate are in very close proximity, they behave as if they are part of the same molecule- it gives the reaction intramolecular character- massive rate increases. reactions occur faster if the reaction is intramolecular.

43. 2) ELECTROSTATIC EFFECTS the strength of electrostatic interactions is related to the capacity of surrounding solvent molecules to reduce the attractive forces between chemical groups. Because water is largely excluded from the active sites as the substrate binds, the dielectric constant near the active site is often low, this may influence the chemical reactivity of the substrate. weak electrostatic interactions, such as those between permanent and induced dipoles in both the active site and the substrate are believed to contribute to the catalysis. A more efficient binding of substrate lowers the free energy of transition state- accelerates the reaction.

44. 3) ACID-BASE CATALYSIS Chemical groups can often be made more reactive by adding or removing a proton. Enzyme active sites contain side chain groups that act as proton donors or acceptors. These groups referred as- general acids and general bases (substance that can release proton/accept proton, respectively) General acids/base- release/accept proton and contribute to reaction rate acceleration.

45. 4) COVALENT CATALYSIS In some enzymes a nucleophilic side chain group forms an unstable covalent bond with the substrate The enzyme-substrate complex then forms product The covalent bond must, at a later stage in the reaction, be broken to regenerate the enzyme. This mechanism is found in enzymes such as proteases like chymotrypsin and trypsin, where an acyl-enzyme intermediate is formed.

46. Serine proteases R-C-NH-R’ + Enzyme-CH2OH R-C-O-CH2-Enzyme R-C-OH + Enzyme-CH2OH Serine proteases- Uses the –CH2OH group as a nucleophile to hydrolyze peptide bonds R’-NH2 H2O

47. The role of cofactors in Enzyme catalysis Catalytic activity of some enzymes depends only on interaction between active site amino acids and substrate other enzymes require non protein compounds for their activities (cofactor) Enzyme cofactors- ions such as Mg2+ /Zn2+ , or Complex organic molecules referred to as coenzyme Apoenzyme = protein component of an enzyme that lacks an essential cofactor Holoenzyme = intact enzyme with bond cofactor

49. Cofactors: METALS 2 classes of metals- transition metals (Fe2+ & Cu2+ ) - alkali & alkaline earth metals (Na+, K+, Mg2+ and Ca2+ ) Transition metals- the most often involved in catalysis- electronic structures. Metal ions provide a high concentration of +ve charge, is useful in binding small molecules Transition metals act as lewis acid (electron pair acceptors)- effective electrophiles (amino acid side chains- poor electrophiles- cannot accept unshared pairs of electrons). Because their directed velences allow them to interact with 2/ more ligands, metal ionshelp to orient the substrate within the active site. The substrate-metal ion complex polarizes the substrate and promotes catalysis

50. Coenzymes Coenzymes are derived by vitamins Vitamins- organic nutrients required in small amounts in human diet. 2 classes – water-soluble - lipid-soluble Vitamin-like nutrients (eg. Lipoic acid, carnitine) that can be synthesized in small amounts, facilitate enzyme-catalyzed reaction