Mutations in the B12-Binding Region of Methionine Synthase: How the Protein Controls Methylcobalamin Reactivity
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- 作者：Joseph T. Jarrett ; Mohan Amaratunga ; Catherine L. Drennan ; Jeffrey D. Scholten ; Richard H. Sands ; Martha L. Ludwig ; and Rowena G. Matthews
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：February 20, 1996
- 年：1996
- 卷：35
- 期：7
- 页码：2464 - 2475
- 全文大小：740K
- 年卷期：v.35,no.7(February 20, 1996)
- ISSN：1520-4995
文摘
Vitamin B12-dependent methionine synthasecatalyzes the transfer of a methyl group frommethyltetrahydrofolate to homocysteine via the enzyme-bound cofactormethylcobalamin. To carry outthis reaction, the enzyme must alternately stabilize six-coordinatemethylcobalamin and four-coordinatecob(I)alamin oxidation states. The lower axial ligand tothe cobalt in free methylcobalamin is thedimethylbenzimidazole nucleotide substituent of the corrin ring; whenmethylcobalamin binds to methioninesynthase, this ligand is replaced by histidine 759, which in turn islinked by hydrogen bonds to aspartate757 and thence to serine 810. We have proposed that these residuescontrol the reactivity of the enzyme-bound cofactor both by increasing the coordination strength of theimidazole ligand and by allowingstabilization of cob(I)alamin via protonation of theHis-Asp-Ser triad. In this paper we report results ofmutation studies focusing on these catalytic residues. We haveused visible absorbance spectroscopy andelectron paramagnetic resonance spectroscopy to probe the coordinationstate of the cofactor and haveused stopped-flow kinetic measurements to explore the reactivity ofeach mutant. We show that mutationof histidine 759 blocks turnover, while mutations of aspartate 757 orserine 810 decrease the reactivity ofthe methylcobalamin cofactor. In contrast, we show that mutationsof these same residues increase therate of AdoMet-dependent reactivation of cob(II)alaminenzyme. We propose that the reaction with AdoMetproceeds via a different transition state than the reactions withhomocysteine and methyltetrahydrofolate.These results provide a glimpse at how a protein can control thereactivity of methylcobalamin.