Synthesis, characterization, solution stability, and X-ray crystal structure of the thiolatocobalamin gamma-glutamylcysteinylcobalamin, a dipeptide analogue of glutathionylcobalamin: insights into the enhanced Co-S bond stability of the natural product gl
Synthesis, characterization, solution stability, and X-ray crystal structure of the thiolatocobalamin gamma-glutamylcysteinylcobalamin, a dipeptide analogue of glutathionylcobalamin: insights into the enhanced Co-S bond stability of the natural product gl
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硫醇钴胺γ-谷氨酰半胱氨酰钴胺（谷胱甘肽钴胺素的二肽类似物）的合成、表征、溶液稳定性和 X 射线晶体结构：深入了解天然产物 gl 增强的 Co-S 键稳定性
DOI：
10.1021/ic001365n
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发表时间：
2001
影响因子：
4.6
通讯作者：
Finke,RG
中科院分区：
文献类型：
--
作者：
Suto,RK;Brasch,NE;Anderson,OP;Finke,RG
谷胱甘肽钴胺素（γ-glutamylcysteinylglycinylcobalamin; γ-GluCysGly-Cbl）是一种天然产物，在活性B12辅酶腺苷钴胺素和甲基钴胺素的生物合成中作为中间体发挥作用。本研究关注的是谷胱甘肽钴胺素与其他硫代钴胺素相比的独特稳定性，特别是稳定性低≥6 × 104倍的半胱氨酰钴胺素，Cys-Cbl。为了确定谷胱甘肽三肽的哪些部分有助于谷胱甘肽钴胺素的总体稳定性，使用两种含半胱氨酸的二肽（谷胱甘肽的截短形式）合成其相应的钴胺素，特别是γ-谷氨酰半胱氨酸Cbl（γ-GluCys-Cbl）和半胱氨酸甘氨酰钴胺素（CysGly-Cbl）。与谷胱甘肽Cbl一样，二肽γ-GluCys-Cbl形成稳定的硫代钴胺素。然而，最有趣的是，观察到CysGly-Cbl与Cys-Cbl一样不稳定。结果表明，与半胱氨酰钴胺素及其类似物相比，谷胱甘肽钴胺素及其同类物的额外稳定性必须来自半胱氨酰钴胺素中的γ-NH3+基团的不稳定性，或谷胱甘肽钴胺素中的γ-NHC（O）−酰胺键的稳定性，或两者兼而有之。为了探索含γ-GluCys钴胺素可能稳定的基态结构基础，γ-GluCys-Cbl被结晶，并产生了第一个真硫钴胺素的X射线结构测定，以及含有Co-S键的钴胺素的第二个结构，第一个例子是Randaccio和同事1999年的硫酮复合物，硫脲钴胺素，（NH 2）2CSCbl。γ-谷氨酰半胱氨酰钴胺素结构的主要特征包括：（i）正常Co-S键长为2.267（2）π，（ii）Co-N（轴向）键长为2.049（6）π，（iii）γ-谷氨酰半胱氨酰部分的两种交替构象，（iv）咕啉环向上折叠24.2°，这是钴胺素迄今观察到的最高折叠程度。这些结果没有显示出任何强稳定性（例如，没有缩短的Co−S键），尽管还不清楚拉长的Co−N（轴向）键的作用是什么（稳定还是不稳定）;相反，晶体学结果表明，亚稳态Cys-Cbl可能具有稳定的Co−S裂解过渡态（沿着可能的是Co−S键的基态不稳定）。总的来说，结果强烈表明，在γ-NH3+上放置正电荷稳定了Co−S键断裂过渡态，从而为所需的完整热产物和动力学研究奠定了基础，作为轴向碱基开关平衡的函数，这将需要更详细地了解谷胱甘肽-（γ-谷氨酰半胱氨酰甘氨酰-）和γ-谷氨酰半胱氨酰钴的独特稳定性。
Glutathionylcobalamin (γ-glutamylcysteinylglycinylcobalamin; γ-GluCysGly-Cbl) is a natural product which functions as an intermediate in the biosynthesis of the active B12coenzymes adenosylcobalamin and methylcobalamin. Of interest to the present studies is glutathionylcobalamin's unique stability in comparison to other thiolatocobalamins, notably the ≥6 × 104fold less stable cysteinylcobalamin, Cys-Cbl. In order to determine which parts of the glutathione tripeptide contribute to the overall stability of glutathionylcobalamin, two cysteine-containing dipeptides, which are truncated versions of glutathione, were used to synthesize their corresponding cobalamins, specifically γ-glutamylcysteinylCbl (γ-GluCys-Cbl) and cysteinylglycinylcobalamin (CysGly-Cbl). As with glutathionylCbl,the dipeptideγ-GluCys-Cbl forms a stablethiolatocobalamin.However and most interestingly, CysGly-Cbl isobserved to be unstable much like Cys-Cbl. The results require that the extra stability of glutathionylcobalamin and its congeners, compared to cysteinylcobalamin and its analogues, must be derived fromdestabilizationby the γ-NH3+group in cysteinylcobalamin, orstabilizationby the γ-NHC(O)− amide linkage in glutathionylcobalamin,or both. To probe any ground-state structural basis for the possible stabilization in γ-GluCys-containing cobalamins, γ-GluCys-Cbl was crystallized and yielded the first X-ray structural determination of a truethiolatocobalamin, and only the second structure of a cobalamin containing a Co−S bond, the first example being Randaccio and co-workers' 1999 structure of the thioketone complex, thioureacobalamin, (NH2)2CSCbl. Key features of the structure of γ-glutamylcysteinylcobalamin include (i) a normal Co−S bond length of 2.267(2) Å, (ii) a Co−N(axial) bond length of 2.049(6) Å, (iii) two alternate conformations of the γ-glutamylcysteinyl moiety, and (iv) folding of the corrin ring upward by 24.2°, the highest degree of folding yet observed for a cobalamin. These results do not show any strong stabilization (e.g., no shortened Co−S bond), although it is not clear for certain what the effect is (stabilizing or destabilizing) of the elongated Co−N(axial) bond; instead, the crystallographic results suggest that the metastable Cys-Cbl probably has a Co−S cleavage transition state that is stabilized (along with, possibly, any ground-state destabilization of the Co−S bond). Overall, the results strongly suggest that placing a positive charge on the γ-NH3+stabilizes the Co−S bond cleavage transition state, thereby setting the stage for the needed full thermolysis product and kinetic studiesas a function of the axial-base on−off equilibriumthat will be required to understand in even greater detail the unique stability of glutathionyl- (γ-glutamylcysteinylglycinyl-) and γ-glutamylcysteinylcobalamins.