Characterization of the Zinc Sites in Cobalamin-Independent and Cobalamin-Dependent Methionine Synthase Using Zinc and Selenium X-ray Absorption Spectroscopy
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- 作者：Katrina Peariso ; Zhaohui S. Zhou ; April E. Smith ; Rowena G. Matthews ; and James E. Penner-Hahn
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：January 30, 2001
- 年：2001
- 卷：40
- 期：4
- 页码：987 - 993
- 全文大小：81K
- 年卷期：v.40,no.4(January 30, 2001)
- ISSN：1520-4995
文摘
X-ray absorption spectroscopy has been used to investigate binding of selenohomocysteine tocobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthase enzymes ofEscherichia coli. We have shown previously [Peariso et al. (1998) J. Am. Chem. Soc. 120, 8410-8416]that the Zn sites in both enzymes show an increase in the number of sulfur ligands when homocysteinebinds. The present data provide direct evidence that this change is due to coordination of the substrate tothe Zn. Addition of L-selenohomocysteine to either MetE or the N-terminal fragment of MetH, MetH(2-649), causes changes in the zinc X-ray absorption near-edge structure that are remarkably similar tothose observed following the addition of L-homocysteine. Zinc EXAFS spectra show that the addition ofL-selenohomocysteine changes the coordination environment of the zinc in MetE from 2S + 2(N/O) to2S + 1(N/O) + 1Se and in MetH(2-649) from 3S + 1(N/O) to 3S + 1Se. The Zn-S, Zn-Se, and Se-Sbond distances determined from the zinc and selenium EXAFS data indicate that the zinc sites in substrate-bound MetE and MetH(2-649) both have an approximately tetrahedral geometry. The selenium edgeenergy for selenohomocysteine shifts to higher energy when binding to either methionine synthase enzyme,suggesting that there is a slight decrease in the effective charge of the selenium. Increases in the Zn-Cysbond distances upon selenohomocysteine binding together with identical magnitudes of the shifts to higherenergy in the Se XANES spectra of MetE and MetH(2-649) suggest that the Lewis acidity of the Znsites in these enzymes appears the same to the substrate and is electronically buffered by the Zn-Cysinteraction.