Mutations of endo-beta-N-acetylglucosaminidase H active site residueAs sp130 anG glu132: activities and conformations.
Mutations of endo-beta-N-acetylglucosaminidase H active site residueAs sp130 anG glu132: activities and conformations.
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内切-β-N-乙酰氨基葡萄糖苷酶 H 活性位点残基 As sp130 和 G glu132 的突变：活性和构象。
DOI：
10.1110/ps.8.11.2338
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
VanRoey,P
中科院分区：
文献类型：
--
作者：
Rao,V;Cui,T;Guan,C;VanRoey,P
内切-β-N-乙酰氨基葡萄糖苷酶H水解高甘露糖和杂合天冬酰胺连接寡糖的N，N′-二乙酰壳二糖核心的β-（1 - 4）-糖苷键。七个突变体的活性位点残基，Asp 130和Glu 132，已制备，测定和结晶。它们包括每个残基到相应酰胺、到Ala和到交替酸性残基以及到双酰胺突变体的单位点突变体。Asp 130的突变体比相应的Glu 132突变体更有活性，与后者残基作为主要催化残基的分配一致。酰胺突变体比交替酸性残基突变体更有活性，交替酸性残基突变体又比Ala突变体更有活性。Asp 130的Asn突变体和双突变体的结构与野生型酶的结构非常相似。突变残基周围的几个残基，包括一些形成β-桶核心的残基，特别是Tyr 168和Tyr 244，在Asp 130的其他两个突变体和Glu 132的Asp突变体的结构中采用非常不同的构象。结果表明，β桶上层的残基可以组织成两种非常不同的堆积排列，这取决于微妙的静电和空间差异，并极大地影响底物结合裂缝的几何形状。因此，几种突变体的相对活性由结构变化定义，除了功能变化外，还导致底物结合受损。
Endo‐β‐N‐acetylglucosaminidase H hydrolyzes the β‐(1‐4)‐glycosidic link of theN,N′‐diacetylchitobiose core of high‐mannose and hybrid asparagine‐linked oligosaccharides. Seven mutants of the active site residues, Asp130 and Glu132, have been prepared, assayed, and crystallized. They include single site mutants of each residue to the corresponding amide, to Ala and to the alternate acidic residue, and to the double amide mutant. The mutants of Asp130 are more active than the corresponding Glu132 mutants, consistent with the assignment of the latter residue as the primary catalytic residue. The amide mutants are more active than the alternate acidic residue mutants, which in turn are more active than the Ala mutants. The structures of the Asn mutant of Asp130 and the double mutant are very similar to that of the wild‐type enzyme. Several residues surrounding the mutated residues, including some that form part of the core of the β‐barrel and especially Tyr168 and Tyr244, adopt a very different conformation in the structures of the other two mutants of Asp 130 and in the Asp mutant of Glu132. The results show that the residues in the upper layers of the β‐barrel can organize into two very distinct packing arrangements that depend on subtle electrostatic and steric differences and that greatly affect the geometry of the substrate‐binding cleft. Consequently, the relative activities of several of the mutants are defined by structural changes, leading to impaired substrate binding, in addition to changes in functionality.