Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

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Enzymatic Addition/Elimination Reactions 199 iso-citrate − O 2 C EnzS H Fe S O H S Fe S Fe Fe S EnzS SEnz CO − 2 H* CO 2 − H O − BEnz Ser 642 − O 2 C EnzS Fe S OH S Fe S Fe Fe S EnzS SEnz cis-aconitate CO − 2 *H CO 2 − O Ser 642 ‘flipping’ of cis-aconitate EnzS Fe S S S Fe Fe EnzS citrate H CO − 2 O H H* CO − 2 HO Fe CO − 2 S SEnz Ser 642 EnzS Fe S OH S Fe S Fe Fe S EnzS SEnz H CO − 2 O *H CO − 2 CO − 2 BEnz Ser 642 Figure 8.10 Mechanism for aconitase. role of the iron–sulphur cluster is to function as a Lewis acid group to facilitate C2O cleavage during the catalytic mechanism. Subsequently, the activation of water as iron(II) hydroxide could provide a reactive nucleophile for water addition. Examination of the enzyme crystal structure indicated that Ser-642 is well positioned as the base for proton abstraction of isocitrate. The stereochemical course of both citrate and isocitrate dehydrations has been shown to be anti-, thus an E2 mechanism is likely as shown in Figure 8.10. It has also been shown that the proton abstracted by Ser-642 is returned upon hydration of cis-aconitate (although the hydroxyl group removed is exchanged). This implies both that Ser- 642 is somehow shielded from solvent water and that the intermediate cisaconitate is Xipped over in the active site before rehydration. This brief discussion covers by no means all of the types of hydratase enzymes found in biological systems, but illustrates a few of the most common examples, and the types of mechanistic and stereochemical methods used in other cases. 8.3 Ammonia lyases The enzymatic elimination of ammonia is carried out on several l-amino acids in biological systems: l-phenylalanine, l-tyrosine, l-histidine and l-aspartic acid, as shown in Figure 8.11. In each of these cases the C b 2H bond being
200 Chapter 8 *H R H H CO 2 − NH 3 + R = Ph R = imidazole R = CO 2 H R CO 2 − + NH 4 + phenylalanine ammonia lyase histidine ammonia lyase aspartase Figure 8.11 Reactions catalysed by some ammonia lyases. broken lies adjacent to an activating group in the b-position of the amino acid side chain. The histidine and phenylalanine ammonia lyases utilise a novel mechanism to assist the leaving group properties of ammonia, which would normally be a poor leaving group. These enzymes are readily inactivated by treatment with nucleophilic reagents, implying that there is an electrophilic group present at the enzyme active site. Examination of puriWed enzyme revealed that the electrophilic cofactor has a characteristic ultraviolet (UV) absorption at 340 nm. Inactivation of phenylalanine ammonia lyase with NaB 3 H 4 followed by acidic hydrolysis of the protein gave 3 H-alanine, suggesting that the electrophilic group might be a dehydroalanine amino acid residue, formed by dehydration of a serine residue. However, determination of the X-ray crystal structure of Pseudomonas putida histidine ammonia lyase in 1999 revealed that the electrophilic prosthetic group is 3,5-dihydro-5-methylene-imidazolone, formed by dehydration of Ser-143, and cyclisation of the amide nitrogen of Gly-144 with the amide carbonyl of Ala-142, as shown in Figure 8.12. Substitution of Ser-143 for threonine using site-directed mutagenesis gives an inactive enzyme; however, a Cys-143 mutant enzyme is able to form the dehydroalanine cofactor by elimination of hydrogen sulphide. Two possible mechanisms for the phenylalanine ammonia lyase reaction are illustrated in Figure 8.13. Attack of the a-amino group of the substrate upon the methylene group of the cofactor (path A), followed by 1,3-prototropic shift, generates a secondary amine intermediate, in which the amine is activated as an enamide leaving group. Anti-elimination of this intermediate leaves the amine covalently attached to the cofactor. Prototropic shift, followed by elimination of ammonia, regenerates the methylene–imidazolone cofactor. NH 142 O 144 HN HN 143 O H N O NH N N O H N O OH Figure 8.12 Methylene-imidazolone cofactor.
Page 1 and 2:
Introduction to Enzyme and Coenzyme
Page 4 and 5:
Introduction to Enzyme and Coenzyme
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Contents Preface Representation of
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Contents vii 8 Enzymatic Addition/E
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Representation of Protein Three-Dim
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2 Chapter 1 H 2 N O NH 2 + H 2 O Ja
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4 Chapter 1 Table 1.1 The vitamins.
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6 Chapter 1 has very diVerent biolo
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2 All Enzymes are Proteins 2.1 Intr
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10 Chapter 2 (Gln). There are three
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12 Chapter 2 AAA Lys ACA Thr AGA Ar
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14 Chapter 2 H N O H N O Figure 2.8
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16 Chapter 2 (a) (b) Figure 2.12 St
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18 Chapter 2 Figure 2.14 Structure
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20 Chapter 2 (a) X Y Enzyme + Subst
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22 Chapter 2 maintaining protein te
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24 Chapter 2 surface glycoprotein g
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26 Chapter 2 O-linked glycosylation
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28 Chapter 2 Metalloproteins I. Ber
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30 Chapter 3 O N H R CO 2 H acylase
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32 Chapter 3 (a) Free energy uncata
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34 Chapter 3 Ester k rel Effective
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36 Chapter 3 3.4 The importance of
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38 Chapter 3 pKa Tyrosine CH 2 O H
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40 Chapter 3 glycoside hydrolysis (
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42 Chapter 3 O O Glu 143 O − R H
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44 Chapter 3 the hydroxyl groups of
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46 Chapter 3 E + S E + S ES' ES ‘
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48 Chapter 3 Examination of protein
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50 Chapter 3 (Note: Similar results
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52 Chapter 4 (1) Direct UV (2) Radi
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54 Chapter 4 Figure 4.3 PuriWcation
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56 Chapter 4 The two kinetic consta
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58 Chapter 4 Lineweaver−Burk Plot
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60 Chapter 4 E + S K i EI+ I ES E +
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62 Chapter 4 (2) by treatment with
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64 Chapter 4 In general the stereoc
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H D T CO 2 H CO − 2 T HO D H −
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68 Chapter 4 4.5 The existence of i
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70 Chapter 4 18O 18O 18 O O P O −
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72 Chapter 4 If a reaction involves
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74 Chapter 4 O D H ketosteroid isom
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76 Chapter 4 Table 4.3 Group speciW
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78 Chapter 4 [S] (mM) Rate of produ
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80 Chapter 4 Enzyme kinetics I.H. S
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82 Chapter 5 O = C NHR peptidase H
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84 Chapter 5 non-speciWc protease c
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86 Chapter 5 His 57 His 57 Asp 102
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88 Chapter 5 Other serine proteases
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90 Chapter 5 Figure 5.8 Structure o
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92 Chapter 5 now predominate Perhap
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OH R O Zn 2+ O O O Ph Glu 270 H 2 N
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96 Chapter 5 CASE STUDY: HIV-1 prot
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98 Chapter 5 HO Transition state pe
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100 Chapter 5 The aminoacyl group o
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102 Chapter 5 5.5 Enzymatic phospho
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Page 119 and 120:
108 Chapter 5 Adenosine 5 0 -tripho
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110 Chapter 5 functional group. Gly
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112 Chapter 5 CH 2 OH O RO HO OH O
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114 Chapter 5 to these atoms that t
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116 Chapter 5 Problems (1) Using pa
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118 Chapter 5 (6) Retaining glycosi
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120 Chapter 5 J.R. Knowles (1980) E
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122 Chapter 6 +1.0 Redox potential
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Table 6.1 Classes of redox enzymes.
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126 Chapter 6 An important point is
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128 Chapter 6 O H − OH − O OH H
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130 Chapter 6 one-electron transfer
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132 Chapter 6 (a) R N H + N O R N H
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134 Chapter 6 Inactivation by trany
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138 Chapter 6 (a) (b) Figure 6.23 S
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140 Chapter 6 glutathione called tr
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142 Chapter 6 neither has a stable
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144 Chapter 6 of the haem cofactor
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146 Chapter 6 Figure 6.33 Active si
Page 159 and 160: 148 Chapter 6 HO H N N O O H N prol
Page 161 and 162: 150 Chapter 6 R R R O 2 , Fe 2+ O O
Page 163 and 164: 152 Chapter 6 CoA. Explain these re
Page 165 and 166: 154 Chapter 6 NADH models O. Almars
Page 167 and 168: 7 Enzymatic Carbon-Carbon Bond Form
Page 169 and 170: 158 Chapter 7 O H − OH O − O H
Page 171 and 172: 160 Chapter 7 Figure 7.4 Structure
Page 173 and 174: 162 Chapter 7 Figure 7.6 Active sit
Page 175 and 176: 164 Chapter 7 7.3 Claisen enzymes I
Page 177 and 178: 166 Chapter 7 CoAS O EnzB − H D T
Page 179 and 180: 168 Chapter 7 onto the acetyl-thioe
Page 181 and 182: 170 Chapter 7 In the case of erythr
Page 183 and 184: 172 Chapter 7 O HO O − O ATP ADP
Page 185 and 186: 174 Chapter 7 CO 2 − vitamin K-de
Page 187 and 188: 176 Chapter 7 7.8 Thiamine pyrophos
Page 189 and 190: 178 Chapter 7 HN N + N H NH S O OPP
Page 191 and 192: 180 Chapter 7 O OH OH camphor geran
Page 193 and 194: 182 Chapter 7 CH 3 * OPP (−)pinen
Page 195 and 196: 184 Chapter 7 Figure 7.36 Structure
Page 197 and 198: 186 Chapter 7 C C C C C O OCH 3 C H
Page 199 and 200: 188 Chapter 7 AcO HO HO NHAc O OH +
Page 201 and 202: 190 Chapter 7 (9) Usnic acid is a n
Page 203 and 204: 192 Chapter 7 Radical couplings W.M
Page 205 and 206: 194 Chapter 8 C-O cleavage H E1 H C
Page 207 and 208: 196 Chapter 8 leads to the formatio
Page 209: 198 Chapter 8 aconitase citrate cis
Page 213 and 214: 202 Chapter 8 EnzB − 2 H − O 2
Page 215 and 216: 204 Chapter 8 involves no change in
Page 217 and 218: 206 Chapter 8 Glyphosate H H N CO
Page 219 and 220: 208 Chapter 8 (5) Chorismic acid (s
Page 221 and 222: 9 Enzymatic Transformations of Amin
Page 223 and 224: CO − − 2 CO 2 H H CO 2 − N +
Page 225 and 226: 214 Chapter 9 - BEnz CO − 2 Cl H
Page 227 and 228: 216 Chapter 9 Arg 292 Arg 292 Lys 2
Page 229 and 230: 218 Chapter 9 Arg 292 Asp 292 HN H
Page 231 and 232: 220 Chapter 9 S − B 1 Enz H − C
Page 233 and 234: 222 Chapter 9 9.6 N-Pyruvoyl-depend
Page 235 and 236: 224 Chapter 9 The biosyntheses of n
Page 237 and 238: 226 Chapter 9 Further reading Gener
Page 239 and 240: 228 Chapter 10 B 1 - H + NH 3 CO
Page 241 and 242: 230 Chapter 10 ‘low-barrier’ +
Page 243 and 244: 232 Chapter 10 H S C 6 H 13 HN His
Page 245 and 246: 234 Chapter 10 O H NH 3 CO 2 − CO
Page 247 and 248: 236 Chapter 10 sub-units. Each sub-
Page 249 and 250: 238 Chapter 10 Further reading Gene
Page 251 and 252: 11 Radicals in Enzyme Catalysis 11.
Page 253 and 254: 242 Chapter 11 CH 2 Co III Ad H OH
Page 255 and 256: 244 Chapter 11 − O 2 C − O CO
Page 257 and 258: 246 Chapter 11 H N H O 734 H N H PF
Page 259 and 260: 248 Chapter 11 H H* + H + H 3 N CO
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250 Chapter 11 O HN NH + H 3 N H 3
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252 Chapter 11 cofactor is involved
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254 Chapter 11 Protein Radicals J.
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256 Chapter 12 self-splicing reacti
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258 Chapter 12 Figure 12.2 Structur
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260 Chapter 12 antigen combining si
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262 Chapter 12 R O OR' OH − R O
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264 Chapter 12 CO 2 − − O 2 C O
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266 Chapter 12 (1) Selective substr
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268 Chapter 12 HO O HO OH HO OH O O
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270 Chapter 12 Problems (1) How rev
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Appendix 1: Cahn-Ingold-Prelog Rule
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Appendix 2: Amino Acid Abbreviation
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276 Appendix 3 Preparation of orang
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278 Appendix 4 (2) Intramolecular a
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280 Appendix 4 from water at a-posi
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282 Appendix 4 Chapter 8 (1) Treat
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284 Appendix 4 (b) Formation of rad
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286 Index b-hydroxydecanoyl thioest
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288 Index glycosylation 26-7 glysop
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290 Index phenylalanine ammonia lya
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292 Index transition states 31-2 an
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