Structure and activity of an active site substitution of ricin A chain

Philip J. Day, Stephen R. Ernst, Arthur E. Frankel, Arthur F. Monzingo, John M. Pascal, Maria C. Molina-Svinth, Jon D. Robertus

Research output: Contribution to journalArticle

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Abstract

The A chain of' ricin (RTA) is an N-glycosidase which inactivates ribosomes by removing a single adenine base from a conserved region of rRNA. X-ray structures and site-directed mutagenesis revealed that Arg 180 interacts will the target adenine hydrogen bonding with N3. It may fully or partially protonate that atom as part of the hydrolysis mechanism. Arg 180 was previously converted to His (RI80H) and shown to greatly reduce activity. Here RI80H is shown to reduce overall activity 500-fold against Artemia Salina ribosomes. A 2.2 Å crystal structure reveals the mutation causes a rearrangement of the active site cleft, with Tyr 80 moving to block access to the adenine recognition site. His 180 forms a strong aromatic interaction with Trp 211, Tyr 80, and Tyr 123. A complex is formed with 250 mM AMP. The nucleotide binds in the active site region, but in an apparently nonproductive orientation. His 180 cannot bond to N3 and is screened from the substrate analog by the intervening Tyr 80. It may be that natural polynucleotide substrates, using additional interactions, can displace Tyr 80 and effect a productive binding.

Original languageEnglish (US)
Pages (from-to)11098-11103
Number of pages6
JournalBiochemistry
Volume35
Issue number34
DOIs
StatePublished - 1996

Fingerprint

Ricin
Adenine
Catalytic Domain
Substitution reactions
Ribosomes
Artemia
Polynucleotides
Mutagenesis
Rapid thermal annealing
Glycoside Hydrolases
Substrates
Adenosine Monophosphate
Hydrogen Bonding
Site-Directed Mutagenesis
Hydrolysis
Hydrogen bonds
Nucleotides
Crystal structure
X-Rays
X rays

ASJC Scopus subject areas

  • Biochemistry

Cite this

Day, P. J., Ernst, S. R., Frankel, A. E., Monzingo, A. F., Pascal, J. M., Molina-Svinth, M. C., & Robertus, J. D. (1996). Structure and activity of an active site substitution of ricin A chain. Biochemistry, 35(34), 11098-11103. https://doi.org/10.1021/bi960880n

Structure and activity of an active site substitution of ricin A chain. / Day, Philip J.; Ernst, Stephen R.; Frankel, Arthur E.; Monzingo, Arthur F.; Pascal, John M.; Molina-Svinth, Maria C.; Robertus, Jon D.

In: Biochemistry, Vol. 35, No. 34, 1996, p. 11098-11103.

Research output: Contribution to journalArticle

Day, PJ, Ernst, SR, Frankel, AE, Monzingo, AF, Pascal, JM, Molina-Svinth, MC & Robertus, JD 1996, 'Structure and activity of an active site substitution of ricin A chain', Biochemistry, vol. 35, no. 34, pp. 11098-11103. https://doi.org/10.1021/bi960880n
Day PJ, Ernst SR, Frankel AE, Monzingo AF, Pascal JM, Molina-Svinth MC et al. Structure and activity of an active site substitution of ricin A chain. Biochemistry. 1996;35(34):11098-11103. https://doi.org/10.1021/bi960880n
Day, Philip J. ; Ernst, Stephen R. ; Frankel, Arthur E. ; Monzingo, Arthur F. ; Pascal, John M. ; Molina-Svinth, Maria C. ; Robertus, Jon D. / Structure and activity of an active site substitution of ricin A chain. In: Biochemistry. 1996 ; Vol. 35, No. 34. pp. 11098-11103.
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