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

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cob(II)alamin NADPH + H + MSR cob(II)alamin NADPH + H + MSR cob(II)alamin NADPH + H + MSR NADP + NADP + NADP + 88 Scheme 1: Cob(I)alamin is released in solution and diffuses to the ATR cob(I)alamin ATP ATR Scheme 2: MSR reduces ATR-bound cob(II)alamin ATR + cob(II)alamin ATR—cob(II)alamin ATR MSR tight tight complex complex NADPH NADPH NADPH + H + + ATP AdoCbl + PPPi NADP + NADP + NADP + AdoCbl + PPPi + MSR + ATR Figure 3-5. Two schemes depicting the conversion of cob(II)alamin to AdoCbl by the MSR-ATR system. In scheme 1, MSR reduces cob(II)alamin to cob(I)alamin and releases it free in solution for diffusion to the ATR. This scheme seems unlikely due to the high nucleophilic nature of cob(I)alamin. In scheme 2, MSR reduces cob(II)alamin while bound to the ATR thereby sequestering the highly reactive cob(I)alamin molecule and protecting it from oxidation and then adenosylation can occur. MSR, a dual flavoprotein, mediates the one electron transfer to cob(II)alamin from the two-electron donor, NADPH.
Wavelength, (525 nm) 0.24 0.23 0.22 0.21 89 ATR addition 0.20 0 5 10 15 20 Time, (min) Figure 3-6. MSR produces little cob(I)alamin in the absence of the ATR enzyme. During the first phase of the reaction little cob(II)alamin was reduced to cob(I)alamin. During this phase, iodoacetate was used to detect cob(I)alamin. Iodoacetate reacts chemically and quantitatively with cob(I)alamin to form CMCbl and this reaction proceeds with an increase in absorbance at 525 nm. At the 10 min time point, 10 µg of ATR was added to the assay mixture and the rate of cob(I)alamin production increased approximately 46-fold. This large increase in the rate of cob(I)alamin formation suggested a physical interaction between the MSR and ATR enzymes. The assay conditions used were similar to those for the MSR-ATR assay and controls showed that iodoacetate worked effectively under these conditions.
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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
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1997). Both cases of the cblD disor
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23 The cblB complementation group i
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(Watkins et al. 2002). Some patient
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Chapter 2 of this dissertation repo
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Corrin Ring Dimethyl benzimidazole
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Classes Eliminases Dehydratases OH
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A) B) CH 3THF 33 MS-cob(I)alamin Me
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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: Absorbance 0.5 0.4 0.3 0.2 0.1 0.0
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.
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BIOGRAPHICAL SKETCH Nicole Aurora L
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