B12 METABOLISM IN HUMANS By NICOLE AURORA LEAL A ...

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H3C C CO SCoA H 2 propionyl-CoA carboxylase HOOC propionyl-CoA CH 3 CH CO Biotin, HCO 3 2- , ATP, Mg 2+ SCoA (2S)-methylmalonyl-CoA methylmalonyl-CoA epimerase HOOC (2R)-methylmalonyl-CoA methylmalonyl-CoA mutase HOOC C C H 2 H C CO SCoA CH 3 H 2 succinyl-CoA CENTRAL <strong>METABOLISM</strong> adenosylcobalamin CO SCoA 32 Valine Isoleucine Methionine Threonine Odd chain fatty acids Thymine Cholesterol HOOC adenosyltransferase CH 3 CH COOH methylmalonic acid cob(I)alamin cob(II)alamin cob(II)alamin reductase cob(III)alamin reductase GSCbl β-ligand transferase HOCbl Figure 1-3. Pathways of propionyl-CoA catabolism and cobalamin metabolism. Propionyl-CoA carboxylase catalyzes the formation of (2S)-methylmalonyl-CoA. Methylmalonyl-CoA epimerase catalyzes the conversion of (2S)-methylmalonyl-CoA to the (2R) isomer. AdoCbl-dependent methylmalonyl-CoA mutase catalyzes the conversion of (2R)-methylmalonyl-CoA to succinyl-CoA. In humans, a deficiency in MCM or in any of the metabolic steps needed for AdoCbl synthesis leads to methylmalonic aciduria.
A) B) CH 3THF 33 MS-cob(I)alamin Methionine MS-CH3Cbl Synthase MS-cob(II)alamin methionine Methionine Synthase Reductase THF homocysteine MS-CH 3Cbl SAM SAH Figure 1-4. Pathways involving methionine synthase and the reductive activation of methionine synthase by methionine synthase reductase. A) Proposed pathway of methionine synthesis. In this pathway, CH3Cbl-dependent MS transfers a methyl from CH3THF to homocysteine forming methionine. B) Reductive activation of MS-bound cob(II)alamin. MSR reduces cobalamin, and SAM provides a methyl for the reactivation of MS which reenters the methylation cycle.
Page 1 and 2: B12 METABOLISM IN HUMANS By NICOLE
Page 3 and 4: The work in this dissertation is de
Page 5 and 6: TABLE OF CONTENTS Page ACKNOWLEDGME
Page 7 and 8: General Molecular and Protein Metho
Page 9 and 10: LIST OF TABLES Table page 1-1. Aden
Page 11 and 12: 3-7. Stoichiometry of the MSR-ATR s
Page 13 and 14: DTT dithiothreitol E. coli Escheric
Page 15 and 16: Abstract of Dissertation Presented
Page 17 and 18: CHAPTER 1 INTRODUCTION History of C
Page 19 and 20: 3 (DMB). The DMB moiety is covalent
Page 21 and 22: 5 and deoxyadenosine. After hydroge
Page 23 and 24: 7 homocysteine recycling and methio
Page 25 and 26: methylmalonic acid. Methylmalonic a
Page 27 and 28: folate-dependent reactions includin
Page 29 and 30: tetanomorphum, P. shermanii, Euglen
Page 31 and 32: enzyme. This suggests that cob(II)a
Page 33 and 34: at the 5’-C of ATP by cob(I)alami
Page 35 and 36: iochemical studies and complementat
Page 37 and 38: 1997). Both cases of the cblD disor
Page 39 and 40: 23 The cblB complementation group i
Page 41 and 42: (Watkins et al. 2002). Some patient
Page 43 and 44: Chapter 2 of this dissertation repo
Page 45 and 46: Corrin Ring Dimethyl benzimidazole
Page 47: Classes Eliminases Dehydratases OH
Page 51 and 52: propanol dehydrogenase (PduQ) propa
Page 53 and 54: or from mutations that impair the a
Page 55 and 56: 1989). 5-bromo-4-chloro-3-indolyl-
Page 57 and 58: 41 5’-GCCGCCGGTACCGATGACGACGACAAG
Page 59 and 60: 43 Growth of Adenosyltransferase Ex
Page 61 and 62: anti-rabbit IgG (γ-chain specific)
Page 63 and 64: sequence similarity to a known ATR
Page 65 and 66: ATRs were produced as fusion protei
Page 67 and 68: 51 of the lacI repressor (not shown
Page 69 and 70: in which proteins bind B12, the "ba
Page 71 and 72: libraries may be particularly usefu
Page 73 and 74: 57 Figure 2-1. Multiple sequence al
Page 75 and 76: Table 2-2. Specific activities of b
Page 77 and 78: 61 1 2 3 4 5 25 kDa Figure 2-4. Wes
Page 79 and 80: 63 (cyanocobalamin, CNCbl) and hydr
Page 81 and 82: 65 5’- triphosphate (ATP), and di
Page 83 and 84: extracts of ATR 239K (160 mg protei
Page 85 and 86: Milford, MA). Samples (200 µl) wer
Page 87 and 88: Linearity of the ATR reaction 71 Th
Page 89 and 90: 73 reactions was characteristic of
Page 91 and 92: 75 MSR Produces little Cob(I)alamin
Page 93 and 94: Johnson et al. 2001, Saridakis et a
Page 95 and 96: AdoCbl by the MSR-ATR system. Exper
Page 97 and 98: propionyl-CoA PCC (2S)-methylmalony
Page 99 and 100:
kDa 97 66 45 31 21 14 7 83 1 2 3 4
Page 101 and 102:
85 Table 3-3. The MSR-ATR system se
Page 103 and 104:
Absorbance 0.5 0.4 0.3 0.2 0.1 0.0
Page 105 and 106:
Wavelength, (525 nm) 0.24 0.23 0.22
Page 107 and 108:
Activity, (nmol min-1 Activity, (nm
Page 109 and 110:
al. 1988). The aldehyde is then dis
Page 111 and 112:
CoA-acylating propionaldehyde dehyd
Page 113 and 114:
97 previously published DNA sequenc
Page 115 and 116:
mM CHES (2-[N-cyclohexylamino]ethan
Page 117 and 118:
101 For the conjugation step, strai
Page 119 and 120:
103 from New England Biolabs (Bever
Page 121 and 122:
105 Propionaldehyde Dehydrogenase A
Page 123 and 124:
107 fraction. Following centrifugat
Page 125 and 126:
109 antiserum obtained was specific
Page 127 and 128:
111 biochemical evidence was presen
Page 129 and 130:
113 Table 4-1. Bacterial strains Sp
Page 131 and 132:
115 Table 4-3. Propionaldehyde dehy
Page 133 and 134:
kDa 209 80 49 35 29 117 1 2 3 4 Fig
Page 135 and 136:
CHAPTER 5 CONCLUSIONS In humans, co
Page 137 and 138:
121 substitutions that are common p
Page 139 and 140:
123 Studies also indicated that hum
Page 141 and 142:
LIST OF REFERENCES Aberg, A., S. Ha
Page 143 and 144:
127 Bradford, M. 1976. A rapid and
Page 145 and 146:
129 Fenton, W. A. and L. E. Rosenbe
Page 147 and 148:
131 Johnson, C. L. V. J., E. Pechon
Page 149 and 150:
133 Mellman, I., H. F. Willard and
Page 151 and 152:
135 Rossi, M., J. P. Glusker, L. Ra
Page 153 and 154:
137 Vlasie, M., S. Chowdhury and R.
Page 155 and 156:
BIOGRAPHICAL SKETCH Nicole Aurora L
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