1. Chapter 5 Enzymes Jia-Qing Zhang Ph.D Biochemistry department Medical school Jinan University Mar. 2007

2. What are enzymes? Enzymes are proteins which act as catalysts Catalyst : A catalyst is something which by its very nature increases the rate of a reaction and remain uncharged at the end of reaction.

3. 3 catalyst enzyme

4. 4 Enzymes control and regulate the various metabolic activities inside living cells.

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6. In the absence of an enzyme:

7. whereas in its presence the rate can be increased up to 10 7 fold.

9. The definition of enzymes? Enzymes are powerful and highly effectual biocatalyst produced by living tissues which increase the rate of reactions that occur in the tissue.

10. What are enzymes made up of ? Almost all enzymes are make up of proteins

12. In 1982s, Thomas Cech discovered RNA possessing catalytic activity, were called ribozymes. In 1995, Jack W. Szostak lab reported a DNA fragment with ligase function, termed Deoxyribozyme So biocatalysts have enzyme, ribozymes, deoxyribozyme

13. 13 Section 5.1 Structure and function of Enzymes 5.1.1 Structure of Enzymes 5.1.1.1 Composition of Enzymes Molecules Enzymes are claasified into Simple enzyme (e.g. urease, protease, lipase etc.) Conjugated enzyme. Most enzymes are conjugated. Simple enzymes are all proteins,

14. Conjugated enzymes include protein component and Non protein constituents, Holoenzyme = apoenzyme + cofactor An anpoenzyme is the protein part

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16. 16 Cofactors commonly seen are 1. Metal ions ; iron, magnesium, cobalt manganese Metal ions tightly link to enzyme are known as metalloenzyme those link between E and S(substrate) are called metal activated enzyme(Table 5-2). 2. Low M.W. organic compounds. Vitamins often play their roles in H + ， electron and other chemicals transfer when they take place in metabolism as coenzyme （ Tab5-1 ）

17. 17 Cofactor is divided into: Coenzyme Prosthetic group

18. 18 Differences between coenzyme and prosthetic group 1. Coenzyme linked to apoenzyme loosly and can be aparted from holoenzyme by dialysing,while prosthetic group linked tightly and can not be separated that way.

19. 19 2. Coenzyme leave apoenzyme after catalyzing a reaction, Prosthetic group can not depart from holoenzyme.

20. 20 5.1.1.2 Monomeric enzymes, Oligomeric enzymes and Multienzyme complex

21. 21 monomeric sarcosine oxidase Monomeric enzymes Monomeric enzymes only contains tertiary structure trypsin

22. 22 Oligomeric enzyme Oligomeric enzymes contains two or more polypeptide chains associated by noncovalent forces.

23. 23 Multienzyme complex is that different enzymes catalyze sequential reactions in the same pathway are bound together. PDCPDC Multienzyme complex

24. 24 5.1.2 Active site of an enzyme Active center: the region of an enzyme that contains some chemical groups for binding substrate and for catalyzing the conversion of substrates to products. All groups in active site are termed the essential groups. Essential group: some chemical groups connected to activity of enzyme.They are classified into binding group and catalytic group.

25. 25 底 物底 物 活性中心以外 的必需基团 结合基团 催化基团 活性中心 目 录目 录

27. Active sites are usually located in clefts between domains or subunits or indentations on surface of proteins

28. 28 5.1.3 Structure and function of enzymes 5.1.3.1 The primary structure of enzymes and Its function Simliar catalyzed function means structure homology. eg. Chymotrypsin trypsin and elastase

29. 29 Chymotypsin and trypsin

30. 30 5.1.3.2 The spatial structure of enzymes and its function The catalyzed activity of enzymes also depends on the conformation of enzymes.

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32. Section 5-2 Nomenclature and Classification of Enzyme In Naming an enzyme substrates are stated first ending – ase is affixed Enzymes are grouped into six classes according to the nature of the reactions

35. 4) Peroxidases 5) reductase

42. Catalyze cleavege of bonds by addition of water

45. Catalyxe racemization of optical or geometric Isomers and intramolecular oxidation-reduction reactions

47. Ligases are usually referred to as synthetases

48. Carboxylase

49. 5.3 Properities and catalytic mechanisms of enzymes 5.3.1 properities of enzyme catalytic reactions 5.3.2 catalytic mechanisms of enzymes

50. 50 Properities of enzyme catalytic reactions 1).High catalytic activity of enzymes: (a) Enzymes can decrease activation energy i.e. molecules are activated using activation energy. By decreasing activation energy,enzymes promotes chemical reactions (fig 5-5).

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52. 52 (b) After Product has formed, free Enzymes are released and reused. (c) Enzyme can make a reaction 10 3 -10 7 times faster than uncatalyzed,

53. 53 2) Highly specificity of enzymes: Specificity refers to the ability of an enzyme to discriminate between two competing substrates. The specificity can be divided into 3 types: (a)Absolute specificity: the extreme selectivity of E that allows it to catalyze only a single S. e.g chymotrypsin hydrolysis peptide bond having aromatic AA, trypsin catalyse those having basic AA.

54. 54 (b) Group specificity(relative specificity): Enzymes atc on a group of related substrates e.g. Hexokinase catalyzes the phosphorylation of glucose, mannose, fructose, glucosamine Esterase catalyse the break down of ester bonds.

55. 55 (c) Optical(stereo) specificity: e.g. LDH hydrolyzes L-lactic acid, but not D- lactic acid

56. 56 3). Under control :enzyme can be regulated Regulated in different levels: biosynthesis, allosteric regulation covalent modification isoenzymes.

57. 57 5.3.2 catalytic mechanisms of enzymes Enzymes increase the rate of reaction by lowering the activation energy? Then, how to decrease the energy?

58. 58 Formation of ES complex and Induced-fit hypothesis The enzyme reversibly combines with its substrate to form an ES complex, that subsequently break down to product, regenerating the free enzyme ； E + S ----- ES ------- P + E P

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60. 60 Lock and Key Theory: The specific action of an enzyme with a single substrate can be explained using a Lock and Key analogy first postulated in 1894 by Emil Fischer. In this analogy, the lock is the enzyme and the key is the substrate. Only the correctly sized key (substrate) fits into the key hole (active site) of the lock (enzyme).

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63. 63 The following experimental evidence show the lock and key theory is rigid model and can not explain the ES complex well. For this reason a modification called the induced-fit theory has been proposed

64. 64 Induced Fit Theory: The induced-fit theory assumes that the substrate plays a role in determining the final shape of the enzyme and that the enzyme is partially flexible. II

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66. 66 summaryenzyme\enzyme_binding.swfenzyme\enzyme_binding.swf

67. 67 5. 3.2.3 several factors contribute to enzyme catalysis 2. Electrostatic effects 3. Acid-base catalysis 4. Covalent Catalysis 1. Proximity effects and orientation arrange:

68. 68 1. Proximity effects and orientation arrange: Chemical reactions are based on molecular collision,proximity and orientation lead to correct direction for collision.

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71. 71 2. Electrostatic effects The strength of electrostatic interactions can reduce the attractive forces and increase the chemical reactivity of the substrate. 3. Acid-base catalysis Enzymes is usually ampholyte, can provide acid or basic enviorment, partial proton tranfer from an acid or to a base lowers the activation energy. the rate of reaction can often be promoted by adding or removing a proton.

73. 4. Covalent Catalysis Accelerates reaction rates through the transient formation of a catalyst-substrate covalent bond

75. Section 5.4 Kinetics of Enzyme- catalyzed reactions Kinetics The branch of chemistry that is concerned with the rates of change in the concentration of reactants in a chemical reaction. Enzyme kinetics Study of the rate of change of reactants to products and factors by which rate of reaction is influenced, such as substrates, activators, inhibitors, pH and temperature. 75

76. Enzyme activity can be expressed by kinetic properties: Internation units(IU) one IU is defined as the amount of enzyme that produces 1umole of product per minute. 1 katal(kat) 1 katal(kat) is equal to the amount of enzyme that converts one mole of substrate to product per second.

77. 5.4.1 The effect of substrate on the rate of enzyme-catalyzed reactions

78. 5.4.1.1 The basic conditions for discussing the effect of substrate on the rate of enzyme- catalyzed reactions

79. 79 5.4.1.2 Rectangular hyperbola plot of initial velocity V 0 versus substrate concentration [S]

81. [S] V Vmax At low [s], V 0 increases linearly with increase in [S]

83. [S] V Vmax At higher [S], V 0 increases nonlinearly with increase in [S]

84. [S] V Vmax At very higher [S], V 0 increases is vanishingly small with increase in [S]

86. 5.4.1.3 Formulation of the Michaelis- menten equation Michaelis-menten equation, a mathematical equation expressing the hyperbolic relationship between the initial velocity, V o, and the substrate concentration, [S].

90. The enzymes-catalyzed reaction process may be summarized as follow: k2k2 k3k3 k4k4,so k 4 ingored

91. k2k2 k3k3

92. k3k3 k3k3 k2k2 [1] k3k3

93. 93 k3k3 k3k3 k2k2 [1] At this point, it ’ s important to draw your attention to two assumption mentioned above: one is [s] >>[E]. the other is, it ’ s assumed that the system is in steady state, that the ES complex is being formed and broken down at the same rate. So that overall [ES] is constant.

94. 94 Formation equal to breakdown, then K1[E][S] = (k2 + k3) [ES] The formation of ES will depend on the k1 and the availability of E and S. So Rate of ES formation = k1 [E][S] The breakdown of [ES] can occur in two ways, either the conversion of S to P, or non-reactive dissociation of S from ES complex. Rate of ES breakdown = (k2 + k3) [ES]

95. 95 [ES] k1 K2 + k3 = [ E][S] By rearranging K1[E][S] = (k2 + k3) [ES] [2]

96. 96 [ES] = [ E][S] As the name implies, these 3 rate terms k are constants, so Michaelis actually combine them into one term, this new constant is termed the michaelis constant and is written Km k1 K2 + k3 = Km Substituting the Km into equation [2] [2] Km [ES] k1 K2 + k3 = [ E][S]

97. 97 The total amount of enzyme in the system must be the same throughout the experiment, but it can either be free (unbound) E or in complex with substrate, ES. If we term the total enzyme E t, this relationship can be written out: t [3] Substituting this definition of [E] back into equation [3] gives us:

98. 98 t t [E] = [Et] – [ES]

99. 99 First of all, open the bracket so that the [E] and [ES] are separately multiplied by [S] t Next, multiply each side by K M, this gives us: t t

100. 100 Then collect the two [ES] terms together on the same side. This gives: t Then because both terms on the right-hand side are multiplied by [ES] we can collect them together into a bracket: Dividing both sides by (K M + [S]) now gives us: t t

101. 101 t Substituting this left-hand side into Equation 1 in place of [ES] results in: 3 t 0 [4]

102. 102 The maximum rate, which we can call V max, would be achieved when all of the enzyme molecules have substrate bound. Under conditions when [S] is much greater than [E], it is fair to assume that all E will be in the form ES. Therefore [Et] = [ES] Notice that k 3 [Et] was present in equation 4, so we can replace this with Vmax, giving a final equation: Thinking again about Equation 1, we could substitute the term V max for v and [E t ] for [ES]. This would give us: Vmax = k 3 [Et]

103. 103 0 This final equation is actually called the Michaelis - Menten equation. 3 t 0 [4]

104. 104 5.4.1.4 The Significance of Km and Vmax Let us consider the case when V is exactly half of V max. Under those circumstances, the Michaelis- Menten equation could be written: On dividing both sides by V max this becomes:

105. 105 Multiplying both sides by (KM + [S]) gives: And then multiplying both sides by 2 further resolves the equation to:

106. 106 [S] on the right-hand side is the same as [S] + [S], so we can take away one [S] from each side. Thus when the rate of the reaction is half of the maximum rate: If we now reconsider the graph that present at the start of this class it could be written:

107. 107 Significance of Km: 1.Km is an important constant depends on E, S structure and reaction environment. 2. Km is equal to the [S] at which the reaction occurs at half of the Vmax rate.

108. 108 3. Sometime(When k 2 is much greater than k 3 ), km can indicates the affinity of the ES complex. Some enzymes have more than 2 substrates, higher Km, weaker binding of S and E. e.g. Glucose and Fructose are substrates of hexokinase, Glu has less Km, higher affinity to hexokinase, in contrast, Fru. has higher Km and less affinity. If E has more than 2 substrates, the least Km is the optimum

109. Significance of Vmax: Vmax, the maximal initial velocity, is obtained only when all the enzymes is in the form of the ES complex, from which it follows that: From above equation, Vmax = k3 [Et] 3 2 3 2

110. 110 K 3 is the turnover number of enzymes. Turnover number of an enzyme is the number of substrate molecules converted into product by an enzyme molecule in a unit of time when the enzyme is fully saturated with substrate.

111. , Vmax

113. 113 Double reciprocal plot ( Lineweaver-Burk plot) In order to obtain accurate Km, the Michaelis formula can be turned into a linear formula by up- side-down the numerator / denominator so as to obtain 1/ Vmax at y axis and 1/Km at x axis.

114. 0 up-side-down the numerator / denominator, give us

115. 1/v 0 : y 1/[s]: x

119. 119 The temperature at which enzymes operate at maximal efficiency Optimum temperature

122. 122 At a pH above this optimum, the enzyme's activity will be reduced and therefore the reaction rate will be lowered; at a pH below this optimum, the enzyme's activity again will be reduced and lower reaction rates result

124. 5.4.5 The effect of inhibitors on the rate of enzyme-catalyzed reactions Inhibitor (I) An agent that can decrease enzyme activity without causing denaturation of enzyme. Inhibitor can be classified into: (1)Irreversible inhibitors: (2) Reversible inhibitors: Competitive, un- competitive, non-competitive

125. Irreversible inhibition Irreversible inhibitors bond and destroy a functional group in an enzyme that is essential for the enzyme activity

126. 126 In most cases forms a covalent link with the enzyme Permanently renders the protein inactive

127. 127 AChE Inhibitor--Organic phosphorus Inhibition of AChE by organic phosphorus PAM Organic phosphorus (public Hazards in agricultural drugs) : usually binds to active site of acetylcholine esterase. ACh

128. -noncompetitive

129. 129 Chemicals that resemble an enzyme's normal substrate and compete with it for the active site. Block active site from the substrate. If reversible, the effect of these inhibitors can be overcome by increased substrate concentration. Competitive inhibition

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132. 132 In competitive inhibition, both inhibitor and substrate can bind to enzyme and form two independent complexes. Only ES degrades to products: EI is considered a 'dead-end'. Because the inhibitor binds to the active site, the substrate cannot (and vice versa), so there cannot be an ternary ESI complex.

133. 133 Mechanism and Features: 1. I and S compete the active site; 2. Which one occupies the active site depends on the affinity of S / I affinity to E and concentrations between I and S. 3. Km increase ( affinity decrease), Vmax unchanged

134. 134

135. Mathematical analysis For those who are interested in Maths only

136. 136 km

137. Noncompetitive inhibition Noncompetitive inhibitors :Enzyme inhibitors that do not enter the enzyme's active site, but bind to another part of the enzyme molecule. Causes enzyme to change its shape so the active site cannot bind substrate.

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141. 141 The Vmax is decreased without a change in the Km for the substrate The inhibition cannot be overcome by increaseing the substrate concentration. Features of non-competitive inhibition:

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144. 144 Uncompetitive inhibition Uncompetitive inhibitors bind only to the ES complex and not the free enzyme.

147. Features: Decrease of Km and Vmax. summary

148. 148 5.4.6 Activators of enzymes Activator: Agents increase the activity of enzyme or make the inactive form become active form are called Activator of enzyme. They are mostly metal ions(Mg 2+,K +,Mn 2+ ),also some anion(Cl - )and organic compounds. Most anions are necessary to enzyme activity,they are called essensial activator.Of course there are non-essential activator,Cl - is so to ptyalin.

149. Section 5.5 Regulation of Enzyme Activity Allosteric regulation Covalent modification There are two major strategies for regulating enzymes:

151. 151 The mechanism of Allosteric regulation

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153. 153 General properties of allosteric enzymes 1.The activities of allosteric enzymes are charged by metabolic activators and inhibitors, which seldom resemble the substrates or products. So allosteric regulation is not inhibition. 2.Modulators bind noncovalently to enzymes. 3.Allosteric enzymes almost possess quaternary structure. 4.Allosteric enzymes often display sigmoid plots of the the reaction V versus S

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155. 155 Covalent modification The covalent attachment of another molecule can modify the activity of enzymes and many other proteins. In these instances, a donor molecule provides a functional moiety that modifies the properties of the enzyme. Most modifications are reversible.

156. 156 Phosphorylation and dephosphorylation are the most common but not the only means of covalent modification.

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159. 159 Other forms of modification: Acetylation Methylation nucleoside modification. Significance of modification : Rapidly change between the active and inactive form

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161. 161 Zymogen Several enzymes are produced and stored as inactive precursors called zymogens

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166. 166 e.g. LDH(lactate dehydrogenase) LDH 1,2,3,4,5 are HHHH, HHHM, HHMM, HMMM and MMMM. i.e. LDH isozymes are tetramers formed by 2 sets of subunits.

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168. 168 Significance : In the heart, LDH1 (4 H) H=heart, catalyzes Lactate convert to pyruvate for energy supply In muscles, LDH5 ( 4M ) M=muscle, convert pyr to Lact. For energy storage. Clinical diagnosis using isozyme. E.g. when heart attack(infarction) happens, enzymes release from injured cells to the blood showed different enzyme(isozyme ) pattern. Isozyme pattern: different isozymes appear as a peak sooner or later followed by the progress of the disease.

169. 169 Summary 1.Definition: active site, allosteric regulation, Covalent modification, Zymogen, Isoenzymes, Coenzyme, prothetic group, 2. Explain the kinetic significance of Km and Vmax 3. What would affect the activity of an enzyme, and how? 4. What are differences betweeen competitive, noncometitive and uncompetitive inhibition? 6. How does the value of Km and Vmax change when adding competitive, noncompetitive and uncompetitive inhibitor respectively?