XRAY DIFFRACTION OPERATIONS ON X9B

我们检查了MS的突变形式，以检查His的作用 (759)、Asp（757）和Ser（810）残基的氢键作用 网络 Co（II）的X射线边缘结果（与EPR数据一致） MS突变体H759G的一种形式表明，用Gly取代His（759） 残基导致四配位物种的形成（鉴定为 通过1s-4pz转换，没有Gly连接到Co。 显示了催化活性的五个数量级的降低， 野生型Co（II）（马修斯等人，生物化学，已提交）。 这一点得到了我们先前的结果的支持，即四配位Co（II） 类咕啉种类非常不稳定（维尔特等人，J. Am.化学会， 1993，115，5299和Scheuring等人，J. Phys. Chem.，1996年，100， 3344）。 EXAFS的结果也支持一个四配位的物种， 平均Co-Neq距离为1.89 +/-0.01 E，与其他钴胺素相似 Hspecies. 突变体D757N的Co（II）形式，其中Asp（757）是 H被Glu替换，表示与 野生型HCo（II）。 EPR数据显示，该突变体是70%的bas-off， 它的H活性被削弱了50倍。 这些结果表明， (759)在HMS中的催化活性和任何变化是必不可少的， H氢键网络显著降低活性。

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.