1. Chapter 3 - Enzymes

2. Outline

3. Introduction to Enzymes Thousands of biochemical reactions proceed at any given instant within living cells. These reactions are catalyzed by enzymes; Enzymes are specific, versatile, and very effective biological catalysts, resulting in much higher reaction rates as compared to chemically catalyzed reactions under ambient conditions. Enzymes are usually proteins of high molecular weight (15000<MW< several million Daltons) that acts as catalysts. (Dalton: one twelfth of the mass of an unbound neutral atom of carbon 12) Enzymes play key functions in controlling rate of reaction, coupling reactions, and sensing the momentary metabolic needs of the cell.

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5. Introduction to Enzymes Urease is a nickel-containing enzyme that catalyzes the hydrolysis of urea to form ammonia and carbamine acid. The latter compound spontaneously decomposes to generate a second molecule of ammonia and carbon dioxide.

6. Introduction to Enzymes Holoenzyme = apoenzyme + cofactor (Enzyme plus a nonprotein group) (protein part) (e.g. metal ions, Mg, Zn, Mn, Fe) (e.g. complex organic molecule - NAD, FAD, CoA, or some vitamins)

7. EC 1 Oxidoreductases: catalyze oxidation/reduction reactionsOxidoreductasesoxidation EC 2 Transferases: transfer a functional group (e.g. a methyl or phosphate group)Transferasesfunctional group EC 3 Hydrolases: catalyze the hydrolysis of various bondsHydrolaseshydrolysis EC 4 Lyases: cleave various bonds by means other than hydrolysis and oxidationLyases EC 5 Isomerases: catalyze isomerization changes within a single moleculeIsomerasesisomerization EC 6 Ligases: join two molecules with covalent bondsLigasescovalent bonds Enzyme Classification - According to the reaction they catalyze

8. Enzyme Commission Number (E.C.) The assignment of E.C. numbers is described in guidelines set out by the International Union of Biochemistry and Molecular Biology, and follows the format E.C. w.x.y.z, where numerical values are substituted for w, x, y and z. The value of w is always between 1 and 6, and indicates one of six main divisions. The value of x indicate the subclassification (p 58, table 3.1). The value of y indicate a subsubclassification, related to more specific information; The value of z is the number for further identification.

9. EC1 OxidoreductasesOxidoreductases http://www.chem.qmul.ac.uk/iubmb/enzyme

10. EC2 TransferasesTransferases

11. EC3 HydrolasesHydrolases

12. EC4 LyasesLyases

13. EC5 IsomerasesIsomerases

14. EC6 LigasesLigases EC 6.5EC 6.5 Forming Phosphoric Ester Bonds EC 6.5.1.1EC 6.5.1.1 DNA ligase (ATP)

15. EC 1.1.3.4 Glucose oxidase EC 1 Oxidoreductases EC 1.1 Acting on the CH-OH group of donors EC 1.1.3 With oxygen as acceptor EC 1.1.3.4 Glucose oxidase (EC 1.1.3.3 malate oxidase and EC 1.1.3.5 hexose oxidase)EC 1.1.3.3EC 1.1.3.5 http://www.chem.qmul.ac.uk/iubmb/enzyme

16. Enzymatic units: Amount of enzyme needed to get a certain level of activity (rate of reaction -  mol product/unit time ) under some controlled conditions. (eg. One unit would be formation of one  mol product per minute at a specific pH and temperature with excessive substrate.) The specific activity is the number of units of activity per amount of total protein (eg. 2 units/mg protein) Originally developed because extractions are ‘dirty’, and hard to guarantee all enzymes are functioning ones. Genetically engineered enzymes can be more concentrated and pure. How to measure how good the enzyme is?

17. Introduction to Enzyme Kinetics Kinetics concerns with the rates of chemical reaction. Enzyme kinetics addresses the biological roles of enzymatic catalysts and quantify the remarkable function of biological enzymes; Enzyme kinetics information can be exploited to control and manipulate the course of metabolic events. Pharmaceuticals or drugs are often special inhibitors targeted at a particular enzyme. Thus the science of pharmacology relies on such information.

18. e.g. HIV protease inhibitor

19. e.g. Penicillin Catalyze cross-linking of bacterial cell wall

20. The Rate Law of Unimolecular reactions Consider a reaction of overall stoichiometry, The rate, or velocity, v of this reaction is the amount of P formed or the amount of A consumed per unit time. Thus: Rate law states that: Where k is rate constant. v is a function of [A] to the first power, or the first order. k is called first order constant.

21. The Rate Law of Bimolecular Reactions Consider a reaction of overall stoichiometry, The rate, or velocity, v of this reaction is the amount of P or Q formed or the amount of A or B consumed per unit time. Thus: Rate law states that: Where k is rate constant. v is a function of [A][B], or second order. k is the second order rate constant.

22. Rate Constant and Free Energy of Activation Arrhenius Equation: Enzymes do not alter the potential of a chemical reaction (i.e., Gibbs free energy). They accelerate the rate of reaction. In another word, they lower the activation energy. Enzyme can accelerate the rate of a reaction by as much as 10 16.

23. How Enzymes Work? T=293 K Van der Waals forces and hydrogen bonding

24. Multisubstrate enzyme catalyzed reactions Proximity effect Enzymes can hold substrates such that reactive regions of substrates are close to each other and to the enzyme’s active site. Orientation effect Enzyme may hold the substrates at certain positions and angles to improve the reaction rate.

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27. Enzyme Kinetics The rate of unimolecular reaction is proportional to the concentration of the reactant. Thus rate is linearily dependent on [A]. A P But if this reaction is catalyzed by an enzyme, the rate shows saturation behavior. Why? v [A] v

28. This is the complete chemical formula for an enzyme-catalyzed (E) reaction of substrate, S and product, P. k -1 K1K1 K -2 K2K2 Enzyme Kinetics

29. k1k1 K -1 k2k2 Initial Velocity Assumption In the beginning of the reaction, there is very little product, or [P] is small. So the amount of [ES] contributed by E+P is negligible. Thus, we concerns the reaction rate that is measured during early reaction period. In which case, the enzyme catalyzed reaction can be modified to: K -1 k1k1 k2k2 K -2

30. Simple Enzyme Kinetics

31. Rapid equilibrium assumption ; The rate of the reverse reaction of second step is negligible as the ES complex is established rather rapidly (proposed by Henri in 1902, Michaelis and Menten in 1913). 1)Initial velocity assumption. Mechaelis-Menten equation describes the relationship between reaction rate and substrate concentration. It can explain the saturation behavior in catalyzed reactions as shown in the previous slide. Rapid Equilibrium Assumption

32. Rapid Equilibrium Assumption ( Michaelis-Menten Kinetics) [E 0 ]=[E]+[ES]

33. Michaelis-Menten Kinetics (cont.)

34. Quasi-Steady-State Assumption Quasi-Steady state is defined as the state during which the enzyme-substrate complex, [ES], remains constant, or ( proposed by G.E. Briggs and J.B.S. Haldane ). Pre-steady state is the state during which [ES] builds up, usually very fast; The equation concerns the reaction rate that is measured only when the steady state has reached.

35. Quasi-Steady-State Assumption Initial velocity assumption and Rate law still apply in enzyme catalyzed reactions. The forward velocity, or rate, v f is, The reverse velocity or rate, or the rate of disappearance v d is, At steady state, there is no accumulation of [ES] or d[ES]/dt = 0, thus: or 0= k1k1 K -1 k2k2

36. Derivation of Equation We need one more condition, that is, the total enzyme concentration, [E 0 ] is the sum of that of enzyme-substrate complex, [ES], and that of free enzyme, [E]: At steady state, the forward rate should equal to the reverse rate: Rate of production formation (rate law), v = k 2 [ES]. So:

37. Notes on the M-M Equations The rate of production formation can usually be measured experimentally by monitoring the progress curve of production formation. The maximum rate can be reached at saturating substrate concentration, or when [S] So M-M equation can be re-written as: Enzyme-catalyzed rate is saturated

38. Sample Question Consider the reversible product-formation using the quasi-steady-state approximation. Develop a rate expression and show that: k1k1 K -1 k2k2 K -2

39. Solution

40. Solution (cont.)

41. Review How to name enzyme? The constituents of enzyme How to classify enzyme and which kind of reaction they catalyze? (Oxidoreductase, Transferase, Hydrolase, Lyase, Isomerase, Ligase)Oxidoreductase TransferaseHydrolaseLyaseIsomeraseLigase Enzymatic unit (One unit -- formation of one  mol product per minute at a specific pH and temperature with excessive substrate) and specific activity (the number of units of activity per amount of total protein)

42. Review (cont.) Enzyme kinetics Initial Velocity Assumption- In the beginning of the reaction, there is very little product, or [P] is small. So the amount of [ES] contributed by E+P is negligible.

43. Review (cont.) Rapid Equilibrium Assumption- The rate of the reverse reaction of second step is negligible as the ES complex is established rather rapidly, and rapid equilibrium is achieved between the enzyme and substrate to form an [ES] complex

44. Review (cont.) Rapid Equilibrium Assumption

45. Review (cont.) Quasi-Steady-State Assumption - the enzyme- substrate complex, [ES], remains constant. 0=

46. Review (cont.) Quasi-Steady-State Assumption 0=