Garza-Ramos G, Tuena de Gomez-Puyou M, Gomez-Puyou A, Yüksel K U, Gracy R W
Instituto de Fisiologia Celular, Universidad Nacional Autonoma de Mexico, D. F., Mexico.
Biochemistry. 1994 Jun 7;33(22):6960-5. doi: 10.1021/bi00188a027.
The specific deamidation of asparagine-71 of triosephosphate isomerase increases upon substrate binding and catalysis. This deamidation at the dimer interface initiates subunit dissociation, unfolding, and protein degradation. The apparent connection between catalysis and terminal marking supports the concept of "molecular wear and tear", and raises questions related to the molecular events that lead to deamidation. In order to explore this interaction, triosephosphate isomerase was entrapped in reverse micelles with different water contents that support different catalytic rates. Deamidation was quantified for the free enzyme, the enzyme in the presence of substrates, and the enzyme which had been covalently modified at the catalytic center with the substrate analogue 3-chloroacetol phosphate (CAP). Both in water and in reverse micelles of cetyltrimethylammonium with 3% and 6% water, substrate binding enhanced deamidation. Studies of the extent of deamidation at various water concentrations showed that deamidation per catalytic turnover was about 6 and 17 times higher in 6% and 3% water than in 100% water, respectively. The enzyme was also entrapped in micelles formed with toluene, phospholipids, and Triton X-100 to explore the process at much lower water concentrations (e.g., 0.3%). Under these conditions, catalysis was very low, and hardly any deamidation took place. Deamidation of the CAP-labeled enzyme was also markedly diminished. At these low-water conditions, the enzyme exhibited markedly increased thermostability and resistance to hydrolysis of the amide bonds. The data suggest that the rate of deamidation not only is dependent on the number of catalytic events but also is related to the time that asparagine-71 exists in a conformation or solvent environment more favorable for deamidation.
磷酸丙糖异构酶天冬酰胺-71的特异性脱酰胺作用在底物结合和催化时增强。二聚体界面处的这种脱酰胺作用引发亚基解离、解折叠和蛋白质降解。催化作用与末端标记之间明显的联系支持了“分子磨损”的概念，并引发了与导致脱酰胺作用的分子事件相关的问题。为了探究这种相互作用，将磷酸丙糖异构酶包埋在具有不同含水量且支持不同催化速率的反胶束中。对游离酶、存在底物时的酶以及在催化中心用底物类似物3-氯乙酰磷酸（CAP）进行共价修饰的酶的脱酰胺作用进行了定量分析。在水以及十六烷基三甲基铵的3%和6%含水量的反胶束中，底物结合均增强了脱酰胺作用。对不同水浓度下脱酰胺作用程度的研究表明，每催化周转的脱酰胺作用在6%和3%含水量时分别比在100%含水量时高约6倍和17倍。该酶还被包埋在由甲苯、磷脂和 Triton X-100形成的胶束中，以在更低的水浓度（如0.3%）下探究这一过程。在这些条件下，催化作用非常低，几乎没有发生脱酰胺作用。CAP标记酶的脱酰胺作用也明显减弱。在这些低水条件下，该酶表现出显著提高的热稳定性和对酰胺键水解的抗性。数据表明，脱酰胺作用速率不仅取决于催化事件的数量，还与天冬酰胺-71存在于更有利于脱酰胺作用的构象或溶剂环境中的时间有关。
