Kitaoka Y
Chemical and Environmental Technology Laboratory, Kobe Steel, Ltd, Japan.
Eur J Biochem. 1998 Oct 1;257(1):255-62. doi: 10.1046/j.1432-1327.1998.2570255.x.
The A chain of ricin (RA) is a cytotoxic RNA N-glycosidase that inactivates ribosomes by depurination of the adenosine residue at position 4324 in 28S rRNA. Of the 267 amino acids in the protein, 231 could be deleted in one or another of 83 mutants, without the loss of the capacity to catalyze hydrolysis of a single specific nucleotide in rRNA [Morris, K. N. & Wool, I. G. (1992) Proc. Natl Acad. Sci. USA 89, 4869-4873]. Expression of 29 selected deletion mutants of RA in prokaryotic cell-free coupled transcription-translation reactions was carried out and the activities of the mutants were assessed by monitoring depurination of reticulocyte ribosomes. Kinetic analysis of five deletion mutants which retained detectable activity was performed. Deletion of amino acids outside the putative active-site cleft in these mutants significantly affected the catalytic rate rather than the interaction with ribosomes. From these data, the amino acids far from the active-site cleft appeared to be involved in alignment of the key residues for catalysis and interaction with the target tetraloop structure of 28S rRNA.
蓖麻毒素A链（RA）是一种细胞毒性RNA N-糖苷酶，它通过使28S rRNA中第4324位的腺苷残基脱嘌呤来使核糖体失活。在该蛋白质的267个氨基酸中，83个突变体中的一个或另一个可以缺失231个氨基酸，而不会丧失催化rRNA中单个特定核苷酸水解的能力[莫里斯，K.N.和伍尔，I.G.（1992年）美国国家科学院院刊89，4869 - 4873]。在原核无细胞偶联转录 - 翻译反应中进行了29个选定的RA缺失突变体的表达，并通过监测网织红细胞核糖体的脱嘌呤来评估突变体的活性。对五个保留可检测活性的缺失突变体进行了动力学分析。这些突变体中推定活性位点裂隙外的氨基酸缺失显著影响催化速率，而不是与核糖体的相互作用。根据这些数据，远离活性位点裂隙的氨基酸似乎参与了催化关键残基的排列以及与28S rRNA靶四环结构的相互作用。
