DIRECT STRUCTURAL OBSERVATION OF AN ACYL-ENZYME INTERMEDIATE IN THE HYDROLYSIS OF AN ESTER SUBSTRATE BY ELASTASE
DIRECT STRUCTURAL OBSERVATION OF AN ACYL-ENZYME INTERMEDIATE IN THE HYDROLYSIS OF AN ESTER SUBSTRATE BY ELASTASE
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DOI：
10.1021/bi00197a032
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发表时间：
1994-08-09
期刊：
影响因子：
2.9
通讯作者：
RINGE, D
中科院分区：
文献类型：
--
作者：
DING, XC;RASMUSSEN, BF;RINGE, D
用X-射线晶体低温酶学方法测定了结晶酶反应路径上一个具有动力学意义的物种的晶体结构。测定了弹性酶催化N-苯甲氧基-L-丙氨酸对硝基苯酯水解反应中的一种特殊的酰基酶中间体的结构，并根据2.3埃分辨率的X射线衍射数据进行了精炼。差值傅立叶电子密度图清楚地显示了被捕获的酰基酶的电子密度。酰基酶是在-26摄氏度形成的，并在数据收集期间稳定在-55摄氏度，利用了蛋白质动力学中发生的玻璃化转变，发生在-50摄氏度左右。
The method of X-ray crystallographic cryoenzymology has been used to determine the crystal structure of a kinetically significant species on the reaction pathway of a crystalline enzyme. The structure of a specific acyl-enzyme intermediate in the elastase-catalyzed hydrolysis of the N-carbobenzoxy-L-alanine p-nitrophenyl ester has been determined and refined against X-ray diffraction data at 2.3-Angstrom resolution. The difference Fourier electron density map clearly shows electron density for the trapped acyl-enzyme. The acyl-enzyme was formed at -26 degrees C and was stabilized at -55 degrees C during data collection, taking advantage of the glass transition in protein dynamics that occurs at around -50 degrees C.