ANALYSIS OF SEVERAL KEY ACTIVE-SITE RESIDUES OF RICIN-A CHAIN BY MUTAGENESIS AND X-RAY CRYSTALLOGRAPHY
ANALYSIS OF SEVERAL KEY ACTIVE-SITE RESIDUES OF RICIN-A CHAIN BY MUTAGENESIS AND X-RAY CRYSTALLOGRAPHY
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DOI：
10.1093/protein/5.8.775
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发表时间：
1992-12-01
期刊：
影响因子：
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通讯作者：
ROBERTUS, JD
中科院分区：
文献类型：
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作者：
KIM, Y;ROBERTUS, JD
活性位点的蓖麻毒素A链的残基进行了分析，通过定点突变和X-射线衍射，以帮助评估其作用机制，这种有毒的N-糖苷酶。根据X射线研究，Arg 180被认为质子化N3的腺嘌呤底物;这有助于键断裂，对作用机制至关重要。将残余物转化为Gln并测量初始速率数据。突变体的K（m）没有受到显著影响，仅增加2倍。然而，k（cat）降低了约1000倍。这是一个简单的解释，Arg 180是涉及更多的过渡态稳定比底物结合。从X射线模型中已知酪氨酸80和123堆叠在底物腺嘌呤环的两侧。当它们各自转化为丝氨酸时，对卤虫核糖体的总体活性分别降低了160和70倍。这些效应各自比当残基先前转化为苯丙氨酸时大约10倍。获得足够的Tyr 80至Phe突变体的蛋白质以进行X射线分析。与诱变数据一起，结构表明两个活性位点Tyr残基的不变性主要由结构稳定性引起。
Active site residues of ricin A chain were analyzed by site-directed mutagenesis and X-ray diffraction to help assess their roles in the mechanism of action of this toxic N-glycosidase enzyme. Arg180 is thought, from X-ray studies, to protonate the adenine substrate at N3; this facilitates bond cleavage and is crucial to the mechanisms of action. The residue was converted to Gln and initial rate data measured. K(m) for the mutant is not significantly affected, increasing only 2-fold. The k(cat), however, is decreased approximately 1000-fold. This is consistent with a simple interpretation that Arg180 is involved more in transition state stabilization than in substrate binding. Tyrosines 80 and 123 are known from X-ray models to stack on either side of the substrate adenine ring. When they were each converted to serine overall activity was reduced 160- and 70-fold respectively against ribosomes from Artemia salina. These effects are each approximately 10 times greater than when the residues were previously converted to phenylalanines. Sufficient protein for the Tyr80 to Phe mutant was obtained to carry out an X-ray analysis. Together with mutagenesis data, the structure suggests that the invariance of the two active site Tyr residues is largely caused by structural stability.