SUBUNIT ASSEMBLY AND ACTIVE-SITE LOCATION IN THE STRUCTURE OF GLUTAMATE-DEHYDROGENASE
SUBUNIT ASSEMBLY AND ACTIVE-SITE LOCATION IN THE STRUCTURE OF GLUTAMATE-DEHYDROGENASE
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DOI：
10.1002/prot.340120109
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发表时间：
1992-01-01
期刊：
影响因子：
--
通讯作者：
STILLMAN, TJ
中科院分区：
文献类型：
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作者：
BAKER, PJ;BRITTON, KL;STILLMAN, TJ
来自共生梭菌的NAD+-连接的谷氨酸脱氢酶的三维晶体结构已经通过同晶置换和分子平均的组合解决到1.96 A分辨率，并细化到0.227的常规晶体学R因子。这种多聚体酶中的每个亚基被组织成两个由深裂缝分开的结构域。一个结构域指导分子自组装成具有32对称性的六聚体寡聚体。另一个结构域在结构上类似于经典的二核苷酸结合折叠，但其中一条链的方向相反。对酶与NAD+的二元复合物的差示傅立叶分析表明，二核苷酸以延伸的构象与两个结构域之间裂缝深处的烟酰胺部分结合。烟酰胺环的羧基酰胺基团与T209和N240的侧链（在所有六聚体GDH序列中保守的残基）之间的氢键为该环的顺式构象异构体提供了正选择。这导致分子排列，其中烟酰胺环的A面被埋在酶表面上，B面暴露出来，邻近包括K89，K113和K125在内的保守残基的引人注目的簇。与化学修饰数据相关的建模研究表明，该区域为谷氨酸/2-酮戊二酸结合位点，并在分子水平上为GDH在催化循环过程中的氢化物转移的B型立体特异性提供了解释。
The three-dimensional crystal structure of the NAD+-linked glutamate dehydrogenase from Clostridium symbiosum has been solved to 1.96 A resolution by a combination of isomorphous replacement and molecular averaging and refined to a conventional crystallographic R factor of 0.227. Each subunit in this multimeric enzyme is organised into two domains separated by a deep cleft. One domain directs the self-assembly of the molecule into a hexameric oligomer with 32 symmetry. The other domain is structurally similar to the classical dinucleotide binding fold but with the direction of one of the strands reversed. Difference Fourier analysis on the binary complex of the enzyme with NAD+ shows that the dinucleotide is bound in an extended conformation with the nicotinamide moiety deep in the cleft between the two domains. Hydrogen bonds between the carboxyamide group of the nicotinamide ring and the side chains of T209 and N240, residues conserved in all hexameric GDH sequences, provide a positive selection for the syn conformer of this ring. This results in a molecular arrangement in which the A face of the nicotinamide ring is buried against the enzyme surface and the B face is exposed, adjacent to a striking cluster of conserved residues including K89, K113, and K125. Modeling studies, correlated with chemical modification data, have implicated this region as the glutamate/2-oxoglutarate binding site and provide an explanation at the molecular level for the B type stereospecificity of the hydride transfer of GDH during the catalytic cycle.