Use of Ammonia-Substituted Chromium Nucleotides for Micromapping of Enzyme Active Sites

这个项目的目的是告诉哪一部分的 金属离子配位球面向酶， 当MgATP或MgADP联合收割机与酶如 己糖激酶 为了允许这种微映射，研究人员将 制备和表征ATP和ADP的惰性铬络合物 其在配位圈中含有一个或两个氨。 因为氨在配位球中降低了亲和力， 氨与蛋白质接触，但应该 当它面对溶剂时， 这些配合物的常数将告诉金属的哪一部分 离子配位球接触酶，并且其面对酶。 溶剂后 待表征的配合物是：1）12个 单氨铬ATP的异构体和预期的相应 含有ADP的异构体。2)顺式异构体的10种表观异构体 二氨铬ATP和相应的ADP异构体，3） 反式二氨铬ATP或ADP的两种预期异构体。 作为 为了表征这些异构体，他们将试图 使单氨络的两种异构体结晶， 焦磷酸盐，并制备相应的三聚磷酸盐 配合物 他们还将试图结晶其中一种 顺式-焦磷酸二氨铬的对映异构体（其中水作为 以及氨是顺式的）通过使用手性阴离子或阳离子，或 手性溶剂知识的CD光谱的迹象，这些 异构体将有助于表征顺式二氨铬 核苷酸复合物。这些铬络合物，一旦 表征了将用于绘制 己糖激酶，丙酮酸激酶，腺苷酸激酶，和 磷酸果糖激酶，所有这些都接受四水合铬ATP作为 基板。

The purpose of this project is to tell which portion of the metal ion coordination sphere faces the enzyme, and which faces the solvent when MgATP or MgADP combine with an enzyme such as hexokinase. To permit such micromapping the investigators will prepare and characterize inert chromium complexes of ATP and ADP which contain one or two ammonias in the coordination sphere. Because ammonia in the coordination sphere lowers affinity when the ammonia is in contact with the protein, but should have little effect when it faces the solvent, the dissociation constants of these complexes will tell which portion of the metal ion coordination sphere contacts the enzyme, and which faces the solvent. The complexes to be characterized are: 1) the 12 isomers of monoamminechromium ATP and the expected corresponding isomers containing ADP. 2) the apparent 10 isomers of cis- diamminechromium ATP, and the corresponding ADP isomers,3) the two expected isomers of trans-diamminechromium ATP or ADP. As an aid to characterizing these isomers, they will try to crystallize the two isomers of monoamminechromium pyrophosphate,and prepare the corresponding tripolyphosphate complexes. They will also try to crystallize one of the enantiomers of cis-diamminechromium pyrophosphate (where water as well as ammonia is cis) by use of a chiral anion or cation, or a chiral solvent. Knowledge of the signs of the CD spectra of these isomers will help characterize the cis-diamminechromium nucleotide complexes. These chromium complexes, once characterized. will be used to map the active sites of hexokinase, pyruvate kinase, adenylate kinase, and phosphofructokinase, all of which accept tetraaquochromium ATP as a substrate.