Biochemistry

1. Biochemistry Chapter 13: Enzymes Chapter 14: Mechanisms of enzyme action Chapter 15: Enzyme regulation Chapter 17: Metabolism- An overview Chapter 18: Glycolysis Chapter 19: The tricarboxylic acid cycle Chapter 20: Electron transport & oxidative phosphorylation Chapter 22: Gluconeogenesis, glycogen metabolism, and the pentose phosphate pathway 林正輝 612 #5220

2. Chapter 13 Enzymes – Kineticsand Specificity Biochemistry by Reginald Garrett and Charles Grisham

3. What are enzymes, and what do they do? • Biological Catalysts • Increase the velocity of chemical reactions

4. What are enzymes, and what do they do? • Thousands of chemical reactions are proceeding very rapidly at any given instant within all living cells • Virtually all of these reactions are mediated by enzymes--proteins (and occasionally RNA) specialized to catalyze metabolic reactions • Most cells quickly oxidize glucose, producing carbon dioxide and water and releasing lots of energy: C6H12O6 + 6 O2 6 CO2 + 6 H2O + 2870 kJ of energy • It does not occur under just normal conditions • In living systems, enzymes are used to accelerate and control the rates of vitally important biochemical reactions

5. Figure 13.1Reaction profile showing large DG‡ for glucose oxidation, free energy change of -2,870 kJ/mol; catalysts lower DG‡, thereby accelerating rate.

6. Enzymes are the agents of metabolic function • Enzymes form metabolic pathways by which • Nutrient molecules are degraded • Energy is released and converted into metabolically useful forms • Precursors are generated and transformed to create the literally thousands of distinctive biomolecules • Situated at key junctions of metabolic pathways are specialized regulatory enzymes capable of sensing the momentary metabolic needs the cell and adjusting their catalytic rates accordingly

7. Figure 13.2The breakdown of glucose by glycolysis provides a prime example of a metabolic pathway. Ten enzymes mediate the reactions of glycolysis. Enzyme 4, fructose 1,6, biphosphate aldolase, catalyzes the C-C bond- breaking reaction in this pathway.

8. 13.1 – What Characteristic Features Define Enzymes? • Enzymes are remarkably versatile biochemical catalyst that have in common three distinctive features: • Catalyticpower • Specificity • Regulation

9. Catalytic power • Enzymes can accelerate reactions as much as 1016 over uncatalyzed rates! • Urease is a good example: • Catalyzed rate: 3x104/sec • Uncatalyzed rate: 3x10 -10/sec • Ratio is 1x1014 (catalytic power)

10. Specificity • Enzymes selectively recognize proper substances over other molecules • The substances upon which an enzyme acts are traditionally called substrates • Enzymes produce products in very high yields - often much greater than 95%

11. Figure 13.2The breakdown of glucose by glycolysis provides a prime example of a metabolic pathway. Ten enzymes mediate the reactions of glycolysis. Enzyme 4, fructose 1,6, biphosphate aldolase, catalyzes the C-C bond- breaking reaction in this pathway.

12. Figure 13.3 A 90% yield over 10 steps, for example, in a metabolic pathway, gives an overall yield of 35%. Therefore, yields in biological reactions must be substantially greater; otherwise, unwanted by-products would accumulate to unacceptable levels.

13. Specificity • The selective qualities of an enzyme are recognized as its specificity • Specificity is controlled by structureof enzyme • the unique fit of substrate with enzyme controls the selectivity for substrate and the product yield • The specific site on the enzyme where substrate binds and catalysis occurs is called the active site

14. Regulation • Regulation of an enzyme activity is essential to the integration and regulation of metabolism • Because most enzymes are proteins, we can anticipate that the functional attributes of enzymes are due to the remarkable versatility found in protein structure • Enzyme regulation is achieved in a variety of ways, ranging from controls over the amount of enzyme protein produced by the cell to more rapid, reversible interactions of the enzyme with metabolic inhibitors and activators (chapter 15)

15. Nomenclature • Traditionally, enzymes often were named by adding the suffix -ase to the name of the substrate upon which they acted: Urease for the urea-hydrolyzing enzyme or phosphatase for enzymes hydrolyzing phosphoryl groups from organic phosphate compounds • Resemblance to their activity: protease for the proteolytic enzyme • Trypsin and pepsin • International Union of Biochemistry and Molecular Biology (IUBMB) http://www.chem.qmw.ac.uk/iubmb/enzyme/ • Enzymes Commission number: EC #.#.#.#

16. Nomenclature • A series of four number severe to specify a particular enzyme • First number is class (1-6) • Second number is subclass • Third number is sub-subclass • Fourth number is individual entry • For example, ATP:D-glucose-6-phosphotransferase (glucokinase) is listed as EC 2.7.1.2. ATP + D-glucose  ADP + D-glucose-6-phosphate • A phosphate group is transferred from ATP to C-6-OH group of glucose, so the enzyme is a transferase (class 2) • Transferring phosphorus-containing groups is subclass 7 • An alcohol group (-OH) as an acceptor is sub-subclass 1 • Entry 2

17. Classification of protein enzymes • Oxidoreductases catalyze oxidation-reduction reactions • Transferases catalyze transfer of functional groups from one molecule to another • Hydrolases catalyze hydrolysis reactions • Lyases catalyze removal of a group from or addition of a group to a double bond, or other cleavages involving electron rearrangement • Isomerases catalyze intramolecular rearrangement (isomerization reactions) • Ligases catalyze reactions in which two molecules are joined (formation of bonds)

19. EC 2.7.1.1 hexokinaseEC 2.7.1.2 glucokinaseEC 2.7.1.3 ketohexokinaseEC 2.7.1.4 fructokinaseEC 2.7.1.5 rhamnulokinaseEC 2.7.1.6 galactokinase. . . EC 2.7.1.156 adenosylcobinamide kinase

20. Many enzymes require non-protein components called cofactors to aid in catalysis • Coenzymes: many essential vitamins are constituents of coenzyme • Cofactors: metal ions • metalloenzymes • Holoenzyme: apoenzyme (protien) + prosthetic group

22. Other Aspects of Enzymes • Mechanisms - to be covered in Chapter 14 • Regulation - to be covered in Chapter 15 • Coenzymes - to be covered in Chapter 17

23. 13.2 – Can the Rate of an Enzyme-Catalyzed Reaction Be Defined in a Mathematical Way? • Kinetics is concerned with the rates of chemical reactions • Enzyme kinetics addresses the biological roles of enzymatic catalyst and how they accomplish their remarkable feats • In enzyme kinetics, we seek to determine the maximum reaction velocity that the enzyme can attain and its binding affinities for substrates and inhibitors • These information can be exploited to control and manipulate the course of metabolic events

24. Chemical kinetics A  P (A  I  J  P) • rate or velocity (v) v = d[P] / dt or v = -d[A] / dt • The mathematical relationship between reaction rate and concentration of reactant(s) is the rate law v = -d[A] / dt =k[A] • k is the proportional constant or rate constant (the unit of k is sec-1)

25. Chemical kinetics v = -d[A] / dt =k[A] • v is first-order with respect to A The order of this reaction is a first-order reaction • molecularity of a reaction The molecularity of this reaction equal 1 (unimolecular reaction)

26. Figure 13.4Plot of the course of a first-order reaction. The half-time, t1/2, is the time for one-half of the starting amount of A to disappear.

27. Chemical kinetics A + B  P + Q • The molecularity of this reaction equal 2 (bimolecular reaction) • The rate or velocity (v) v = -d[A] / dt = -d[B] / dt = d[P] / dt = d[Q] / dt • The rate law is v = k[A] [B] • The order of this reaction is a second-order reaction • The rate constantk has the unit of M-1 sec-1)

28. The Transition State • Reaction coordinate: a generalized measure of the progress of the reaction • Free energy (G) • Standard statefree energy (25℃, 1 atm, 1 M/each) • Transition state • The transition state represents an intermediate molecular state having a high free energy in the reaction. • Activation energy: • Barriers to chemical reactions occur because a reactant molecule must pass through a high-energy transition state to form products. • This free energy barrier is called theactivation energy.

29. Figure 13.5Energy diagram for a chemical reaction (A→P) and the effects of (a) raising the temperature from T1 to T2 or (b) adding a catalyst. Raising the temperature raises the average energy of A molecules, which increases the population of A molecules having energies equal to the activation energy for the reaction, thereby increasing the reaction rate. In contrast, the average free energy of A molecules remains the same in uncatalyzed versus catalyzed reactions (conducted at the same temperature). The effect of the catalyst is to lower the free energy of activation for the reaction.

30. Decreasing G‡ increase reaction rate Two general ways may accelerate rates of chemical reactions • Raise the temperature The reaction rate are doubled by a 10℃ • Add catalysts • True catalysts participate in the reaction, but are unchangedby it. Therefore, they can continue to catalyze subsequent reactions. • Catalysts change the rates of reactions, but do not affect the equilibrium of a reaction.

31. Most biological catalysts are proteins called enzymes(E). • The substance acted on by an enzyme is called a substrate (S). • Enzymes accelerate reactions by lowering the free energy of activation • Enzymes do this by binding the transition state of the reaction better than the substrate • The mechanism of enzyme action in Chapter 14

32. 13.3 – What Equations Define the Kinetics of Enzyme-Catalyzed Reactions? • The Michaelis-Menten Equation • The Lineweaver-Burk double-reciprocal plot • Hanes-Woolf plot Vmax [S] v = Km + [S]

33. Figure 13.6 A plot of v versus [A] for the unimolecular chemical reaction, A→P, yields a straight line having a slope equal to k.

34. Figure 13.7 Substrate saturation curve for an enzyme-catalyzed reaction. The amount of enzyme is constant, and the velocity of the reaction is determined at various substrate concentrations. The reaction rate, v, as a function of [S] is described by a rectangular hyperbola. At very high [S], v = Vmax. That is, the velocity is limited only by conditions (temperature, pH, ionic strength) and by the amount of enzyme present; v becomes independent of [S]. Such a condition is termed zero-order kinetics. Under zero-order conditions, velocity is directly dependent on [enzyme]. The H2O molecule provides a rough guide to scale. The substrate is bound at the active site of the enzyme.

35. The Michaelis-Menten Equation • Louis Michaelis and Maud Menten's theory • It assumes the formation of an enzyme-substrate complex (ES) E + S ES • At equilibrium k-1 [ES] = k1 [E][S] And Ks = = k1 k-1 [E][S] k-1 [ES] k1

36. The Michaelis-Menten Equation k1 k2 E + S ES E + P • The steady-state assumption ES is formed rapidly from E + S as it disappears by dissociation to generate E + S and reaction to form E + P d[ES] dt • That is; formation of ES = breakdown of ES k1 [E] [S] = k-1[ES] + k2[ES] k-1 = 0

37. Figure 13.8Time course for the consumption of substrate, the formation of product, and the establishment of a steady-state level of the enzyme-substrate [ES] complex for a typical enzyme obeying the Michaelis-Menten, Briggs-Haldane models for enzyme kinetics. The early stage of the time course is shown in greater magnification in the bottom graph.

38. The Michaelis-Menten Equation k1 [E] [S] = k-1[ES] + k2[ES] = (k-1+ k2)[ES] [ES] = ( )[E] [S] Km = Km is Michaelis constant Km [ES] = [E] [S] k1 k-1+ k2 k-1+ k2 k1

39. The Michaelis-Menten Equation Km [ES] = [E] [S] Total enzyme, [ET] = [E] + [ES] [E] = [ET] – [ES] Km [ES] = ([ET] – [ES]) [S] = [ET] [S] – [ES] [S] Km [ES] + [ES] [S] = [ET] [S] (Km + [S]) [ES] = [ET] [S] [ES] = [ET] [S] Km + [S]

40. The Michaelis-Menten Equation [ET] [S] [ES] = The rate of product formation is v = k2 [ES] v = Vmax = k2 [ET] v = Km + [S] k2 [ET] [S] Km + [S] Vmax [S] Km + [S]

41. Understanding Km • The Michaelis constant Km measures the substrate concentration at which the reaction rate is Vmax/2. • Associated with the affinity of enzyme for substrate • Small Km means tight binding; high Km means weak binding

42. v = When v = Vmax / 2 Vmax Vmax [S] 2 Km + [S] Km + [S] = 2 [S] [S] = Km Vmax [S] Km + [S] =

44. Understanding Vmax The theoretical maximal velocity • Vmax is a constant • Vmax is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality • To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate • Vmax is asymptotically approached as substrate is increased

45. The dual nature of the Michaelis-Menten equation Combination of 0-order and 1st-order kinetics • When S is low ([s] <<Km), the equation for rate is 1st order in S • When S is high ([s] >>Km), the equation for rate is 0-order in S • The Michaelis-Menten equation describes a rectangular hyperbolic dependence of v on S • The actual estimation of Vmax and consequently Km is only approximate from each graph

46. Vmax [S] v = Km + [S] When S is low ([s] << Km), Km + [S]=Km When S is high ([s] >>Km), Km + [S]= [S] Vmax [S] v = Km Vmax v =

47. The turnover number A measure of catalytic activity • kcat, the turnover number, is the number of substrate molecules converted to product per enzyme molecule per unit of time, when E is saturated with substrate. • kcat is a measure of its maximal catalytic activity • If the M-M model fits, k2 = kcat = Vmax/Et • Values of kcat range from less than 1/sec to many millions per sec (Table 13.4)

49. The catalytic efficiency Name for kcat/KmAn estimate of "how perfect" the enzyme is • kcat/Kmis an apparent second-order rate constant v = (kcat/Km) [E] [S] • kcat/Km provides an index of the catalytic efficiency of an enzyme • kcat/Km = k1k2/ (k-1 + k2)