Structure and Activity of an Active Site Substitution of Ricin A Chain

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• 作者：Philip J. Day ; Stephen R. Ernst ; Arthur E. Frankel ; Arthur F. Monzingo ; John M. Pascal ; Maria C. Molina-Svinth ; and Jon D. Robertus
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：August 27, 1996
• 年：1996
• 卷：35
• 期：34
• 页码：11098 - 11103
• 全文大小：280K
• 年卷期：v.35,no.34(August 27, 1996)
• ISSN：1520-4995

文摘

The A chain of ricin (RTA) is an N-glycosidase whichinactivates ribosomes by removing asingle adenine base from a conserved region of rRNA. X-raystructures and site-directed mutagenesisrevealed that Arg 180 interacts with the target adenine hydrogenbonding with N3. It may fully or partiallyprotonate that atom as part of the hydrolysis mechanism. Arg 180was previously converted to His (R180H)and shown to greatly reduce activity. Here R180H is shown toreduce overall activity 500-fold againstArtemia salina ribosomes. A 2.2 Å crystal structurereveals the mutation causes a rearrangement of theactive site cleft, with Tyr 80 moving to block access to the adeninerecognition site. His 180 forms astrong aromatic interaction with Trp 211, Tyr 80, and Tyr 123. Acomplex is formed with 250 mMAMP. The nucleotide binds in the active site region, but in anapparently nonproductive orientation. His180 cannot bond to N3 and is screened from the substrate analog by theintervening Tyr 80. It may bethat natural polynucleotide substrates, using additional interactions,can displace Tyr 80 and effect aproductive binding.