XRAY DIFFRACTION OPERATIONS ON X9B

我们已经检查了多发性硬化症的突变形式，以检查His的作用 氢键中的(759)、Asp(757)和Ser(810)残基 网络。Co(II)的X射线边缘结果(与EPR数据一致) MS突变体H759G的形态表明，用甘氨酸取代His(759) 残基导致形成四配位物种(经鉴定 通过1s-4pz的转换而不与Co连接甘氨酸。该突变 显示出催化活性在以下方面下降了五个数量级 至野生型Co(II)(Matthews等人，《生物化学》，提交)。 这得到了我们之前的结果的支持，即一个四坐标的Co(II) 皮质类群物种非常不稳定(Wirt等人，J.Am化学。SOC. 1993,115,5299和Scheuring et al，J.Phys.化学，1996,100， 3344)。EXAFS结果还支持四个坐标物种与 与其他钴胺相似的平均Co-NEQ距离为1.89+/-0.01E 哈桑德。突变体D757N的Co(II)形式，其中Asp(757)是 H替换为Glu，表示与 野生型HCO(II)。EPR数据显示，该突变体的脱落率为70% 它的HActivity被削弱了50倍。这些结果表明，他的 HMS中的(759)对催化活性和 氢键网络大大降低了活性。

We have examined mutant forms of MS to examine the role of the His (759), Asp (757) and Ser (810) residues in the hydrogen bonding network. X-ray edge result (in agreement with EPR data) on the Co(II) form of the MS mutant H759G shows that replacing His (759) with a Gly residue causes the formation of a four-coordinate species (identified by the 1s-4pz transition with no Gly ligation to the Co. This mutant shows five magnitude decrease in the catalytic activity with respect to the wild-type Co(II) (Matthews et al., Biochemistry, submitted). This is supported by our earlier result that a four-coordinate Co(II) corrinoid species is very unstable (Wirt et al., J. Am. Chem. Soc., 1993, 115, 5299 and Scheuring et al, J. Phys. Chem., 1996, 100, 3344). The EXAFS results also support a four-coordinate species with an average Co-Neq distance of 1.89+/-0.01 E similar to other cobalamin Hspecies. The Co(II) form of mutant D757N, in which Asp (757) is Hreplaced by a Glu, indicates similar geometry to that of the wild-type HCo(II). EPR data shows that this mutant is 70% bas-off and its Hactivity is impaired by 50-fold. These results show that His (759) in HMS is essential to the catalytic activity and any change in the Hhydrogen bonding network decreases the activity substantially.