tailieunhanh - Báo cáo khóa học: The effect of mutations surrounding and within the active site on the catalytic activity of ricin A chain

Models for the binding of the sarcin–ricin loop (SRL) of 28S ribosomal RNA to ricinAchain (RTA) suggest that several surface exposed arginine residues surrounding the active site cleft make important interactions with the RNA substrate. The data presented in this study suggest differing roles for these arginyl residues. Substitution of Arg48 or Arg213with Ala lowered the activity of RTA 10-fold. Furthermore, substitutionofArg213withAsp lowered theactivityofRTA 100-fold. The crystal structure of thisRTAvariant showed it to have an unaltered tertiary structure, suggesting that the positively charged state of Arg213 is crucial for activity | Eur. J. Biochem. 271 153-162 2004 FEBS 2003 doi The effect of mutations surrounding and within the active site on the catalytic activity of ricin A chain Catherine J. Marsden Vilmos Fulop Philip J. Day and J. Michael Lord Department of Biological Sciences University of Warwick Coventry UK Models for the binding of the sarcin-ricin loop SRL of 28S ribosomal RNA to ricin A chain RTA suggest that several surface exposed arginine residues surrounding the active site cleft make important interactions with the RNA substrate. The data presented in this study suggest differing roles for these arginyl residues. Substitution of Arg48 or Arg213 with Ala lowered the activity of RTA 10-fold. Furthermore substitution of Arg213 with Asp lowered the activity of RTA 100-fold. The crystal structure of this RTA variant showed it to have an unaltered tertiary structure suggesting that the positively charged state of Arg213 is crucial for activity. Substitution of Arg258 with Ala had no effect on activity although substitution with Asp lowered activity 10-fold. Substitution of Arg134 prevented expression of folded protein suggesting a structural role for this residue. Several models have been proposed for the binding of the SRL to the active site of RTA in which the principal difference lies in the conformation of the second G in the target GAGA motif in the 28S rRNA substrate. In one model the sidechain of Asn122 is proposed to make interactions with this G whereas another model proposes interactions with Asp75 and Asn78. Site-directed mutagenesis of these residues of RTA favours the first of these models as substitution of Asn78 with Ser yielded an RTA variant whose activity was essentially wild-type whereas substitution of Asn122 reduced activity . Substitution of Asp75 failed to yield significant folded protein suggesting a structural role for this residue. Keywords ricin ribosome site-directed mutagenesis heterologous expression. Ricin is