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Methods for Studying Enzymatic Reactions 65 O 2 H P450cam O 2 , NADPH O OH 1 H 1 H RETENTION Figure 4.14 Stereochemistry of P450cam-catalysed reaction. which catalyses the hydroxylation of camphor was shown to proceed with retention of stereochemistry by use of the labelled substrate illustrated in Figure 4.14. Interconversions of methylene (5CH 2 or 2CH 2 2) groups to methyl (2CH 3 ) groups require a special type of stereochemical analysis, since the resulting methyl group contains three apparently identical hydrogen atoms (i.e. an XXXY system). However, it is possible to analyse these methyleneto-methyl interconversions using all three of the isotopes of hydrogen – 1 H, 2 H and 3 H – in the form of a chiral methyl group. It is important to note that in this analysis the 1 Hand 2 H substituents are present in 100% abundance, whereas only a small proportion of molecules contain 3 H (since 3 H is only available and only safe to handle in relatively low abundance). Therefore the analysis of chiral methyl groups must focus on those molecules containing 3 H, by detecting the presence or absence of 3 H label. Chiral methyl groups can be generated from enzymatic reactions by preparing the methylene substrate in a stereospeciWcally labelled form using two of the isotopes of hydrogen, and carrying out the enzymatic reaction in the presence of the third isotope. If the product can be degraded to chiral acetic acid, then the conWguration of the chiral methyl group can be determined using a method developed independently by Cornforth and Arigoni, shown in Figure 4.15. The method of analysis involves conversion to chiral acetyl coenzyme A (CoA) (see Section 5.4), followed by incubation with malate synthase (see Section 7.3), which removes one of the hydrogens on the methyl group, and combines with glyoxalate to form malic acid. The malate synthase reaction has a preference for removal of 1 H rather than 2 Hor 3 H (i.e. a kinetic isotope eVect of k H =k T ¼ 2:7), so in the majority of molecules 1 H is removed. The reaction with glyoxalate then occurs with inversion of conWguration. Therefore, the 2S enantiomer of acetyl CoA is converted into the 2S,3R enantiomer of malate containing 2 H and 3 H stereospeciWcally at C-3, as illustrated in Figure 4.15. Treatment with the enzyme fumarase then results in a stereospeciWc antielimination of water, the enzyme removing only the proR hydrogen at C-3. The conWguration of the major product at C-3 can, therefore, be deduced by
H D T CO 2 H CO − 2 T HO D H − CO 2 fumarase HOD − CO 2 T − CO 2 major product 76.7% retention of 3 H HO H CO 2 − D T fumarase CO 2 − HOT CO 2 − D CO 2 − major product 76.5% loss of 3 H H D stereospecific addition H of tritium to top face D T chemical degradation Analysis of chiral acetic acid: H D T CO 2 − 2R isomer acetate kinase, ATP, Mg 2+ phosphotransacetylase, CoASH H D T O SCoA malate synthase HO 2 C CHO H T D CO 2 − 2S isomer acetate kinase, ATP, Mg 2+ phosphotransacetylase, CoASH H T D O SCoA malate synthase HO 2 C CHO Figure 4.15 Chiral methyl groups. ATP, adenosine triphosphate; CoASH, coenzyme A.
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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
Page 11 and 12:
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
Page 21 and 22:
10 Chapter 2 (Gln). There are three
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12 Chapter 2 AAA Lys ACA Thr AGA Ar
Page 25 and 26: 14 Chapter 2 H N O H N O Figure 2.8
Page 27 and 28: 16 Chapter 2 (a) (b) Figure 2.12 St
Page 29 and 30: 18 Chapter 2 Figure 2.14 Structure
Page 31 and 32: 20 Chapter 2 (a) X Y Enzyme + Subst
Page 33 and 34: 22 Chapter 2 maintaining protein te
Page 35 and 36: 24 Chapter 2 surface glycoprotein g
Page 37 and 38: 26 Chapter 2 O-linked glycosylation
Page 39 and 40: 28 Chapter 2 Metalloproteins I. Ber
Page 41 and 42: 30 Chapter 3 O N H R CO 2 H acylase
Page 43 and 44: 32 Chapter 3 (a) Free energy uncata
Page 45 and 46: 34 Chapter 3 Ester k rel Effective
Page 47 and 48: 36 Chapter 3 3.4 The importance of
Page 49 and 50: 38 Chapter 3 pKa Tyrosine CH 2 O H
Page 51 and 52: 40 Chapter 3 glycoside hydrolysis (
Page 53 and 54: 42 Chapter 3 O O Glu 143 O − R H
Page 55 and 56: 44 Chapter 3 the hydroxyl groups of
Page 57 and 58: 46 Chapter 3 E + S E + S ES' ES ‘
Page 59 and 60: 48 Chapter 3 Examination of protein
Page 61 and 62: 50 Chapter 3 (Note: Similar results
Page 63 and 64: 52 Chapter 4 (1) Direct UV (2) Radi
Page 65 and 66: 54 Chapter 4 Figure 4.3 PuriWcation
Page 67 and 68: 56 Chapter 4 The two kinetic consta
Page 69 and 70: 58 Chapter 4 Lineweaver−Burk Plot
Page 71 and 72: 60 Chapter 4 E + S K i EI+ I ES E +
Page 73 and 74: 62 Chapter 4 (2) by treatment with
Page 75: 64 Chapter 4 In general the stereoc
Page 79 and 80: 68 Chapter 4 4.5 The existence of i
Page 81 and 82: 70 Chapter 4 18O 18O 18 O O P O −
Page 83 and 84: 72 Chapter 4 If a reaction involves
Page 85 and 86: 74 Chapter 4 O D H ketosteroid isom
Page 87 and 88: 76 Chapter 4 Table 4.3 Group speciW
Page 89 and 90: 78 Chapter 4 [S] (mM) Rate of produ
Page 91 and 92: 80 Chapter 4 Enzyme kinetics I.H. S
Page 93 and 94: 82 Chapter 5 O = C NHR peptidase H
Page 95 and 96: 84 Chapter 5 non-speciWc protease c
Page 97 and 98: 86 Chapter 5 His 57 His 57 Asp 102
Page 99 and 100: 88 Chapter 5 Other serine proteases
Page 101 and 102: 90 Chapter 5 Figure 5.8 Structure o
Page 103 and 104: 92 Chapter 5 now predominate Perhap
Page 105 and 106: OH R O Zn 2+ O O O Ph Glu 270 H 2 N
Page 107 and 108: 96 Chapter 5 CASE STUDY: HIV-1 prot
Page 109 and 110: 98 Chapter 5 HO Transition state pe
Page 111 and 112: 100 Chapter 5 The aminoacyl group o
Page 113 and 114: 102 Chapter 5 5.5 Enzymatic phospho
Page 119 and 120: 108 Chapter 5 Adenosine 5 0 -tripho
Page 121 and 122: 110 Chapter 5 functional group. Gly
Page 123 and 124: 112 Chapter 5 CH 2 OH O RO HO OH O
Page 125 and 126: 114 Chapter 5 to these atoms that t
Page 127 and 128:
116 Chapter 5 Problems (1) Using pa
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118 Chapter 5 (6) Retaining glycosi
Page 131 and 132:
120 Chapter 5 J.R. Knowles (1980) E
Page 133 and 134:
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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136 Chapter 6 Figure 6.20 Structure
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138 Chapter 6 (a) (b) Figure 6.23 S
Page 151 and 152:
140 Chapter 6 glutathione called tr
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142 Chapter 6 neither has a stable
Page 155 and 156:
144 Chapter 6 of the haem cofactor
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146 Chapter 6 Figure 6.33 Active si
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148 Chapter 6 HO H N N O O H N prol
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150 Chapter 6 R R R O 2 , Fe 2+ O O
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152 Chapter 6 CoA. Explain these re
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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
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Page 173 and 174:
162 Chapter 7 Figure 7.6 Active sit
Page 175 and 176:
164 Chapter 7 7.3 Claisen enzymes I
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166 Chapter 7 CoAS O EnzB − H D T
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168 Chapter 7 onto the acetyl-thioe
Page 181 and 182:
170 Chapter 7 In the case of erythr
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172 Chapter 7 O HO O − O ATP ADP
Page 185 and 186:
174 Chapter 7 CO 2 − vitamin K-de
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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
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182 Chapter 7 CH 3 * OPP (−)pinen
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Page 197 and 198:
186 Chapter 7 C C C C C O OCH 3 C H
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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 and 210:
198 Chapter 8 aconitase citrate cis
Page 211 and 212:
200 Chapter 8 *H R H H CO 2 − NH
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202 Chapter 8 EnzB − 2 H − O 2
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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
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CO − − 2 CO 2 H H CO 2 − N +
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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
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220 Chapter 9 S − B 1 Enz H − C
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222 Chapter 9 9.6 N-Pyruvoyl-depend
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224 Chapter 9 The biosyntheses of n
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226 Chapter 9 Further reading Gener
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228 Chapter 10 B 1 - H + NH 3 CO
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230 Chapter 10 ‘low-barrier’ +
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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
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236 Chapter 10 sub-units. Each sub-
Page 249 and 250:
238 Chapter 10 Further reading Gene
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11 Radicals in Enzyme Catalysis 11.
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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
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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
Page 289 and 290:
278 Appendix 4 (2) Intramolecular a
Page 291 and 292:
280 Appendix 4 from water at a-posi
Page 293 and 294:
282 Appendix 4 Chapter 8 (1) Treat
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284 Appendix 4 (b) Formation of rad
Page 297 and 298:
286 Index b-hydroxydecanoyl thioest
Page 299 and 300:
288 Index glycosylation 26-7 glysop
Page 301 and 302:
290 Index phenylalanine ammonia lya
Page 303:
292 Index transition states 31-2 an
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