Probing the Role of the Histidine 759 Ligand in Cobalamin-Dependent Methionine Synthase
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- 作者：Matthew D. Liptak ; Angela S. Fleischhacker ; Rowena G. Matthews ; Thomas C. Brunold
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：July 10, 2007
- 年：2007
- 卷：46
- 期：27
- 页码：8024 - 8035
- 全文大小：473K
- 年卷期：v.46,no.27(July 10, 2007)
- ISSN：1520-4995
文摘
Cobalamin-dependent methionine synthase (MetH) of Escherichia coli is a 136 kDa, modularenzyme that undergoes large conformational changes as it uses a cobalamin cofactor as a donor or acceptorin three separate methyl transfer reactions. At different points during the reaction cycle, the coordinationto the cobalt of the cobalamin changes; most notably, the imidazole side chain of His759 that coordinatesto the cobalamin in the "His-on" state can dissociate to produce a "His-off" state. Here, two distinctspecies of the cob(II)alamin-bound His759Gly variant have been identified and separated. Limitedproteolysis with trypsin was employed to demonstrate that the two species differ in protein conformation.Magnetic circular dichroism and electron paramagnetic resonance spectroscopies were used to show thatthe two species also differ with respect to the axial coordination to the central cobalt ion of the cobalamincofactor. One form appears to be in a conformation poised for reductive methylation with adenosylmethionine; this form was readily reduced to cob(I)alamin and subsequently methylated [albeit yielding aunique, five-coordinate methylcob(III)alamin species]. Our spectroscopic data revealed that this formcontains a five-coordinate cob(II)alamin species, with a water molecule as an axial ligand to the cobalt.The other form appears to be in a catalytic conformation and could not be reduced to cob(I)alamin underany of the conditions tested, which precluded conversion to the methylcob(III)alamin state. This formwas found to possess an effectively four-coordinate cob(II)alamin species that has neither water nor histidinecoordinated to the cobalt center. The formation of this four-coordinate cob(II)alamin "dead-end" speciesin the His759Gly variant illustrates the importance of the His759 residue in governing the equilibriabetween the different conformations of MetH.