Effects of specific mutations in active site motifs of 2',5'-oligoadenylate synthetase on enzymatic activity.
2',5'-Oligoadenylate synthetase (2',5'-OAS) is a double-stranded RNA-dependent nucleotidyl-transferase induced by interferon (IFN). Several 2',5'-OAS cDNA have been cloned from human, pig, rat, mouse, and chicken. A P-loop motif followed by an Asp-containing sequence (referred to as D-box) and a region with a high content of Lys and Arg (KR-rich region) are well conserved in 2',5'-OAS. The sequence 196DFLKQR201 of 40-kDa human 2',5'-OAS, to which 8-azido-ATP binds (N. Kon and R.J. Suhadolnik, J. Biol. Chem. 271, 19983-19990, 1996), is included in the KR-rich region. We introduced several site-directed mutations into these active motifs of 42-kDa murine 2',5'-OAS. Each mutant enzyme studied bound to poly(I):poly(C) to the same extent as wild-type enzyme. Both K67R, a P-loop mutant, and K200R, a KR-rich region mutant, exhibited a reduced but considerable rate of enzymatic activities. The activity of the double mutant K67R/K200R was about 10% of the wild type. On the other hand, the activities of both K67M and K200M were not more than 2% of the wild-type enzyme, and no activity was detected in another P-loop mutant, G62A/G63A. The binding of Mg2+ to a D-box mutant D76N/D78N was markedly reduced, and only a very low level of enzymatic activity was detected in this mutant. These results demonstrate that the P-loop, the D-box that binds Mg2+, and the KR-rich region are important for the enzymatic activities of 2',5'-OAS.
Yamamoto
Y
Y
Department of Biotechnology, Kyoto Institute of Technology, Japan.
Sono
D
D
Sokawa
Y
Y
D015088:2',5'-Oligoadenylate Synthetase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D008274:Magnesium / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D011487:Protein Conformation
D015088:2',5'-Oligoadenylate Synthetase; D008274:Magnesium