Importance of the Histidine Ligand to Coenzyme B12 in
the Reaction Catalyzed by Methylmalonyl-CoA Mutase*
Monica
Vlasie,
Shantanu
Chowdhury, and
Ruma
Banerjee
From the Department of Biochemistry, University of Nebraska,
Lincoln, Nebraska 68588-0664
Methylmalonyl-CoA mutase is an adenosylcobalamin
(AdoCbl)-dependent enzyme that catalyzes the rearrangement
of methylmalonyl-CoA to succinyl-CoA. The crystal structure of this
protein revealed that binding of the cofactor is accompanied by a
significant conformational change in which dimethylbenzimidazole, the
lower axial ligand to the cobalt in solution, is replaced by His-610
donated by the active site. The contribution of the lower axial base to
the ~1012-fold rate acceleration of the homolytic
cleavage of the upper axial cobalt-carbon bond has been the subject of
intense scrutiny in the model inorganic literature. In contrast,
trans ligand effects in methylmalonyl-CoA mutase and indeed
the significance of the ligand replacement are poorly understood. In
this study, we have used site-directed mutagenesis to create the H610A
and H610N variants of methylmalonyl-CoA mutase and report that both
mutations exhibit both diminished activity (5,000- and 40,000-fold,
respectively) and profoundly weakened affinity for the native cofactor,
AdoCbl. In contrast, binding of the truncated cofactor analog,
adenosylcobinamide, lacking the nucleotide tail, is less impaired. The
catalytic failure of the His-610 mutants is in marked contrast to the
phenotype of the adenosylcobinamide-GDP reconstituted wild type enzyme
that exhibits only a 4-fold decrease in activity, although His-610 fails to coordinate when this cofactor analog is bound. Together, these
studies suggest that His-610 may: (i) play a structural role in
organizing a high affinity cofactor binding site possibly via
electrostatic interactions with Asp-608 and Lys-604, as
suggested by the crystal structure and (ii) play a role in catalyzing
the displacement of dimethylbenzimidazole thereby facilitating the conformational change that must precede cofactor docking to the mutase
active site.
*
This work was supported by Grant DK45776 from the National
Institutes of Health (to R. B.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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