Sadhu A, Magnuson J A
Biochemistry/Biophysics Program, Washington State University, Pullman 99164-4660.
Biochemistry. 1989 Apr 18;28(8):3197-204. doi: 10.1021/bi00434a013.
The stoichiometry of Mn2+ binding to concanavalin A was found to be influenced by temperature, pH, and the presence or absence of saccharide. Demetalized concanavalin A binds one Mn2+ (S1 site) at 5 degrees C, pH 6.5, and two Mn2+ at 25 degrees C (S1 and S2 sites). The association constants for Mn2+ are 6.2 x 10(5) and 3.7 x 10(4) M-1 for the S1 and S2 sites, respectively, at 25 degrees C. Concanavalin A with one Mn2+ bound per monomer remains in an open conformation and exhibits a relatively high water proton relaxation rate. Concanavalin A with two Mn2+ ions remains in a closed conformation characterized by a lower relaxation rate. The rate of binding of the second Mn2+ to concanavalin A as determined by ESR and the rate of conversion of open form to closed form (folding over) as determined by proton relaxation rate measurements gave an identical rate constant of 80.0 +/- 5.8 M-1 h-1 at 17 degrees C. Ca2+, Sr2+, and high levels of methyl alpha-D-mannopyranoside also induce folding of concanavalin A. Ca2+ is not catalytic but stoichiometric in causing the folding. Mn2+ in the S1 site can be displaced by Ni2+, Co2+, and Zn2+, and Mn2+ in the S2 site can be displaced by Ca2+ and Sr2+. Concanavalin A with Ni2+, Co2+, Zn2+, or Mn2+ in the S1 site and Ca2+ or Sr2+ in the S2 site has a higher affinity for methylumbelliferyl alpha-D-mannopyranoside than Ni-Mn-, Co-Mn-, Zn-Mn-, and Cd-Cd-concanavalin A.
发现锰离子（Mn2+）与伴刀豆球蛋白A的化学计量关系受温度、pH值以及糖类的存在与否影响。脱金属的伴刀豆球蛋白A在5摄氏度、pH值6.5时结合一个Mn2+（S1位点），在25摄氏度时结合两个Mn2+（S1和S2位点）。在25摄氏度时，S1和S2位点与Mn2+的缔合常数分别为6.2×10⁵和3.7×10⁴ M⁻¹。每个单体结合一个Mn2+的伴刀豆球蛋白A保持开放构象，表现出相对较高的水质子弛豫率。结合两个Mn2+离子的伴刀豆球蛋白A保持封闭构象，其特征是弛豫率较低。通过电子自旋共振（ESR）测定的第二个Mn2+与伴刀豆球蛋白A的结合速率，以及通过质子弛豫率测量确定的开放形式向封闭形式的转化速率（折叠），在17摄氏度时给出了相同的速率常数80.0±5.8 M⁻¹ h⁻¹。钙离子（Ca2+）、锶离子（Sr2+）和高水平的α-D-甘露吡喃糖苷甲基也会诱导伴刀豆球蛋白A折叠。Ca2+在导致折叠方面不是催化性的，而是化学计量性的。S1位点的Mn2+可被镍离子（Ni2+）、钴离子（Co2+）和锌离子（Zn2+）取代，S2位点的Mn2+可被Ca2+和Sr2+取代。S1位点含有Ni2+、Co2+、Zn2+或Mn2+且S2位点含有Ca2+或Sr2+的伴刀豆球蛋白A对α-D-甘露吡喃糖苷甲基伞形酮的亲和力高于镍-锰、钴-锰、锌-锰和镉-镉伴刀豆球蛋白A。
