title: Functional role of glutamine 28 and arginine 39 in double stranded RNA cleavage by human pancreatic ribonuclease.
abstract: Human pancreatic ribonuclease (HPR), a member of RNase A superfamily, has a high activity on double stranded (ds) RNA. By virtue of this activity HPR appears to be involved in the host-defense against pathogenic viruses. To delineate the mechanism of dsRNA cleavage by HPR, we have investigated the role of glutamine 28 and arginine 39 of HPR in its activity on dsRNA. A non-basic residue glycine 38, earlier shown to be important for dsRNA cleavage by HPR was also included in the study in the context of glutamine 28 and arginine 39. Nine variants of HPR respectively containing Q28A, Q28L, R39A, G38D, Q28A/R39A, Q28L/R39A, Q28A/G38D, R39A/G38D and Q28A/G38D/R39A mutations were generated and functionally characterized. The far-UV CD-spectral analysis revealed all variants, except R39A, to have structures similar to that of HPR. The catalytic activity of all HPR variants on single stranded RNA substrate was similar to that of HPR, whereas on dsRNA, the catalytic efficiency of all single residue variants, except for the Q28L, was significantly reduced. The dsRNA cleavage activity of R39A/G38D and Q28A/G38D/R39A variants was most drastically reduced to 4% of that of HPR. The variants having reduced dsRNA cleavage activity also had reduction in their dsDNA melting activity and thermal stability. Our results indicate that in HPR both glutamine 28 and arginine 39 are important for the cleavage of dsRNA. Although these residues are not directly involved in catalysis, both arginine 39 and glutamine 28 appear to be facilitating a productive substrate-enzyme interaction during the dsRNA cleavage by HPR.
authors.affiliation: Immunochemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Md Tabish
authors.initials: MT
authors.lastname: Rehman
authors.forename: Punyatirtha
authors.initials: P
authors.lastname: Dey
authors.forename: Md Imtaiyaz
authors.initials: MI
authors.lastname: Hassan
authors.forename: Faizan
authors.initials: F
authors.lastname: Ahmad
authors.forename: Janendra K
authors.initials: JK
authors.lastname: Batra
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D055162:Biocatalysis; D002942:Circular Dichroism; D019295:Computational Biology; D004795:Enzyme Stability; D005973:Glutamine / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011061:Poly A / Q000378:metabolism; D011072:Poly U / Q000378:metabolism; D011489:Protein Denaturation; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D044366:Transition Temperature
chemical_list: D050505:Mutant Proteins; D012330:RNA, Double-Stranded; D005973:Glutamine; D011061:Poly A; D011072:Poly U; D001120:Arginine; D012259:Ribonuclease, Pancreatic