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

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72 6.9 µM, and the Km for cob(I)alamin were 1.2 µM and 1.6 µM, respectively (Figure 3-3). The Vmax values for ATR 239K and 239M were 250 and 200 nmol min -1 mg -1 , respectively. When the Km values for cob(I)alamin were determined, saturating levels of ATP (500 µM) were added to assay mixtures while varying the concentration of cob(I)alamin. Similarly, saturating levels of cob(I)alamin (50 µM) were added to assays when the Km values for ATP were determined. Each value shown in Figure 3-3 is the average of three measurements of the initial reaction rate at the indicated substrate concentrations. MSR Reduces Cob(II)alamin to Cob(I)alamin for AdoCbl Synthesis The human enzyme that catalyzes the third step of CNCbl assimilation, the reduction of cob(II)alamin to cob(I)alamin for AdoCbl synthesis by the ATR enzyme, is unknown. Recently, MSR was shown to catalyze cob(II)alamin reduction for MS activation (Olteanu and Banerjee 2001). To test whether MSR can also reduce cob(II)alamin to cob(I)alamin for AdoCbl synthesis, an in vitro assay was used (MSR-ATR assay). In assays that contained standard components, cob(II)alamin, ATP, DTT, NADPH, 100 µg purified MSR, and 10 µg purified ATR, AdoCbl was formed at a rate of 0.96 nmol min -1 . Controls showed that no measurable AdoCbl was formed in the absence of ATR, MSR, ATP, or cob(II)alamin. When NADPH was excluded from the reaction mixture, 65% of the total activity remained indicating that DTT can partially replace NADPH as the electron donor for this reaction. When DTT was omitted, 100% activity remained with NADPH as the sole electron donor, showing that DTT is not required for the reaction to occur. In addition, several further tests were used to establish that AdoCbl was the product of the reaction. The UV-Visible spectrum of completed
73 reactions was characteristic of AdoCbl, and the reaction product was photolyzed by a 30 min exposure to incandescent light with the formation of cob(II)alamin (Figure 3-4). Moreover, the product of the MSR-ATR reaction co-migrated with authentic AdoCbl by reverse-phase HPLC following co-injection (not shown). These results show that the combination of the MSR and ATR enzymes converted cob(II)alamin to AdoCbl. Since prior studies have shown that the human ATR requires cob(I)alamin for AdoCbl synthesis and is inactive with cob(II)alamin, these results indicate that MSR reduced cob(II)alamin to cob(I)alamin for AdoCbl synthesis by ATR. Cob(I)alamin is Sequestered by the MSR-ATR System Above, we presented evidence that MSR can reduce cob(II)alamin to cob(I)alamin for adenosylation by the ATR. In principle, there are two ways in which this might occur (Figure 3-5). MSR could reduce cob(II)alamin to cob(I)alamin directly, which is subsequently released into solution and diffuses to ATR. Alternatively, MSR could reduce cob(II)alamin bound to ATR and the interaction between the two proteins could sequester cob(I)alamin. To test whether cob(I)alamin was sequestered or released into solution during AdoCbl synthesis by the MSR-ATR system, a chemical trap for cob(I)alamin was used. Iodoacetate (which reacts rapidly and quantitatively with cob(I)alamin to form CMCbl) was added to an MSR-ATR assay at a 1000-fold excess compared to the ATR (400 µM compared to 0.4 µM). The assay was allowed to proceed to completion. Then, CMCbl and AdoCbl were resolved and quantitated by reverse-phase HPLC. Results showed that 35% of the cob(I)alamin formed was converted to CMCbl by reaction with iodoacetate and that 65% was converted to AdoCbl via the ATR enzyme (Table 3-3, line 1). If "free"
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B12 METABOLISM IN HUMANS By NICOLE
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The work in this dissertation is de
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TABLE OF CONTENTS Page ACKNOWLEDGME
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General Molecular and Protein Metho
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LIST OF TABLES Table page 1-1. Aden
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3-7. Stoichiometry of the MSR-ATR s
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DTT dithiothreitol E. coli Escheric
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Abstract of Dissertation Presented
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CHAPTER 1 INTRODUCTION History of C
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3 (DMB). The DMB moiety is covalent
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5 and deoxyadenosine. After hydroge
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7 homocysteine recycling and methio
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methylmalonic acid. Methylmalonic a
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folate-dependent reactions includin
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tetanomorphum, P. shermanii, Euglen
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enzyme. This suggests that cob(II)a
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at the 5’-C of ATP by cob(I)alami
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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 and 48: Classes Eliminases Dehydratases OH
Page 49 and 50: A) B) CH 3THF 33 MS-cob(I)alamin Me
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: Linearity of the ATR reaction 71 Th
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
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123 Studies also indicated that hum
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LIST OF REFERENCES Aberg, A., S. Ha
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127 Bradford, M. 1976. A rapid and
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129 Fenton, W. A. and L. E. Rosenbe
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131 Johnson, C. L. V. J., E. Pechon
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133 Mellman, I., H. F. Willard and
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135 Rossi, M., J. P. Glusker, L. Ra
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137 Vlasie, M., S. Chowdhury and R.
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BIOGRAPHICAL SKETCH Nicole Aurora L
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