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

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cob(I)alamin had been produced under the conditions used, it is expected that the majority should have been converted to CMCbl by reaction with the large excess of iodoacetate prior to diffusion to the ATR for conversion to AdoCbl. Hence, the above 74 results indicated that a significant portion of the cob(I)alamin was sequestered (protected from reaction with iodoacetate) during conversion of cob(II)alamin to AdoCbl by MSR and ATR. As a control, assays were performed that were similar to those described above except that MSR was replaced with the combination of 1 mM DTT plus 50 µM FAD to chemically generate cob(I)alamin in situ. Chemical reduction of cob(II)alamin necessarily produces “free” cob(I)alamin which must then diffuse to the ATR before it can be converted to AdoCbl. Under these conditions, only 10% of the cob(I)alamin formed was converted to AdoCbl while 90% was converted to CMCbl (Table 3-3, line 2). This was in contrast to results obtained when MSR was used to generate cob(I)alamin for the ATR enzyme where 65% of the cob(I)alamin formed was converted to AdoCbl (Table 3-3, line1). Hence these results indicate that the MSR-ATR system sequesters cob(I)alamin during the conversion of cob(II)alamin to AdoCbl. Interestingly, further studies showed that iodoacetate had relatively little effect on the total amount of AdoCbl formed by the MSR-ATR system. In the absence or presence of iodoacetate, 94 or 85 nmol of AdoCbl was produced, respectively (Table 3-3). This indicated that the majority of the cob(I)alamin produced by the MSR-ATR system was sequestered.
75 MSR Produces little Cob(I)alamin in the Absence of the ATR Iodoacetate was also used to test whether MSR can produce “free” cob(I)alamin in the absence of ATR. Assays were prepared that were similar in composition to the MSR-ATR assay except that ATR was replaced by iodoacetate. During the first 10 min of the reaction, cob(I)alamin was formed at a very low rate (0.02 nmol min -1 ) (Figure 3-6). Then, at the 10 min time point, 10 µg of purified ATR was added to the assay and the rate of cob(I)alamin formation increased approximately 46-fold. These findings showed that the presence of the ATR greatly enhanced the production of cob(I)alamin by MSR suggesting a physical interaction between the MSR and ATR. Control experiments showed that BSA had no effect on the rate of cob(I)alamin formation when substituted for ATR in the second phase of the reaction shown in Figure 3-6. Moreover, the controls showed that iodoacetate was working effectively under our assay conditions. In similar reaction mixtures containing DTT and FAD, the cob(I)alamin formed was efficiently trapped by iodoacetate (data not shown). Stoichiometry of the MSR-ATR system The dependence of ATR activity on the molar ratio of MSR/ATR was determined at ratios from 0 to 40 (Figure 3-7). Maximal activity occurred at a ratio of approximately 4 moles MSR (which is a monomer) per mol ATR monomer. This ratio is similar to the optimal stoichiometry for activation of MS by MSR and is a reasonable value for a physiological process (Olteanu and Banerjee 2001). Ionic Strength Dependence of Cobalamin Reduction and Adenosylation by the MSR-ATR System Variation of the ionic strength from 15 to 720 mM had relatively little effect on the synthesis of AdoCbl by the MSR-ATR system (Figure 3-8). Within this range, greater
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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
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 and 88: Linearity of the ATR reaction 71 Th
Page 89: 73 reactions was characteristic of
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
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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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