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

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than 65% activity was retained. This is in contrast to the relatively narrow ionic strength
dependence of MS activation by MSR (Olteanu and Banerjee 2001).
Discussion
Prior studies identified the human ATR and showed that defects in its encoding
gene (MMAB) underlie cblB methylmalonic aciduria (Dobson et al. 2002, Leal et al.
2003). Investigations also identified two common polymorphic variants of the ATR that
are found in normal individuals (Dobson et al. 2002). Here, both ATR variants were
expressed in E. coli, purified, and found to have similar kinetic properties. The specific
activities of variants 239K and 239M were 250 and 200 nmol min -1 mg -1 , and the Km
values were 6.3 and 6.9 µM for ATP and 1.2 and 1.6 µM for cob(I)alamin, respectively.
These values are roughly similar to those previously reported for bacterial ATR enzymes
where specific activities range from 53 to 619 nmol min -1 mg -1 , and Km values from 2.8 to
110 µM for ATP and from 3 to 5.2 µM for cob(I)alamin (Suh and Escalante-Semerena
1995, Johnson et al. 2001, Saridakis et al. 2004). The Km values for the human ATR are
appropriate to its physiological role since cellular levels of cobalamin are typically 1 µM
or less in higher organisms and ATP concentrations are in the low mM range (Schneider
and Stroinski 1987). Thus, the results reported here show that both common ATR
variants are essentially wild-type and have kinetic properties sufficient to meet cellular
needs for AdoCbl synthesis.
Three classes of ATRs that are unrelated in amino acid sequence have been
identified, the PduO-type, CobA-type, and EutT-type (Johnson et al. 2001). Members of
the CobA-type and the PduO-type have been purified, partially characterized, and their
crystal structures determined (Suh and Escalante-Semerena 1995, Bauer et al. 2001,