The differential catalytic activity of ribosome-inactivating proteins saporin 5 and 6 is due to a single substitution at position 162.
Saporin, a type I ribosome-inactivating protein produced by the soapwort plant Saponaria officinalis belongs to a multigene family that encodes its several isoforms. The saporin seed isoform 6 has significantly higher N-glycosidase and cytotoxic activities compared with the seed isoform 5, although the two have identical active sites. In the present study, we have investigated the contribution of non-conservative amino acid changes outside the active sites of these isoforms towards their differential catalytic activity. The saporin 6 residues Lys134, Leu147, Phe149, Asn162, Thr188 and Asp196 were replaced by the corresponding saporin 5 residues, Gln134, Ser147, Ser149, Asp162, Ile188 and Asn196, to generate six variants of saporin 6, K134Q, L147S, F149S, N162D, T188I and D196N. By functional characterization, we show that the change in amino acid Asn162 in saporin 6 to aspartic acid residue of saporin 5 contributes mainly to the lower catalytic activity of saporin 5 compared with saporin 6. The non-involvement of other non-conservative amino acids in the differential catalytic activity of these isoforms was confirmed with the help of the double mutations N162D/K134Q, N162D/L147S, N162D/F149S, N162D/T188I and N162D/D196N.
Ghosh
Paroma
P
Immunochemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Batra
Janendra K
JK
D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D009154:Mutation / Q000235:genetics; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D054789:Ribosome Inactivating Proteins, Type 1; D000076985:Saporins
D010940:Plant Proteins; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D009699:N-Glycosyl Hydrolases; D000076985:Saporins