Human eosinophil-derived neurotoxin: involvement of a putative non-catalytic phosphate-binding subsite in its catalysis.
Human eosinophil-derived neurotoxin (EDN) or RNase 2, found in the non-core matrix of eosinophils is a ribonuclease belonging to the Ribonuclease A superfamily. EDN manifests a number of bioactions including neurotoxic and antiviral activities, which are dependent on its ribonuclease activity. The core of the catalytic site of EDN contains various base and phosphate-binding subsites. Unlike many members of the RNase A superfamily, EDN contains an additional non-catalytic phosphate-binding subsite, P(-1). Although RNase A also contains a P(-1) subsite, the composition of the site in EDN and RNase A is different. In the current study we have generated site-specific mutants to study the role of P(-1) subsite residues Arg(36), Asn(39), and Gln(40) of EDN in its catalytic activity. The individual mutation of Arg(36), Asn (39), and Gln(40) resulted in a reduction in the catalytic activity of EDN on poly(U) and poly(C). However, there was no change in the activities on yeast tRNA and dinucleotide substrates. The study shows that the P(-1) subsite is crucial for the ribonucleolytic activity of EDN on polymeric RNA substrates.
Sikriwal
Deepa
D
Immunochemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
Seth
Divya
D
Dey
Punyatirtha
P
Batra
Janendra K
JK
D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D047208:Eosinophil-Derived Neurotoxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism*; D011071:Poly T / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D011994:Recombinant Proteins; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D010710:Phosphates; D011994:Recombinant Proteins; D011071:Poly T; D004247:DNA; D012343:RNA, Transfer; D047208:Eosinophil-Derived Neurotoxin; D012260:Ribonucleases