Biochemical characterization of a mutant isoleucyl-transfer ribonucleic acid synthetase from Escherichia coli K-12.
Isoleucyl-transfer ribonucleic acid (tRNA) synthetase [l-isoleucine: soluble RNA ligase (adenosine monophosphate), EC 6.1.1.5; IRS] was partially purified from Escherichia coli K-12 and from an ileS mutant that appears to be altered in IRS. The half-life of wild-type IRS, incubated at 60.5 C, is 69 min, whereas that of mutant IRS is 8 min. Mutant IRS shows about a 100-fold lower affinity than wild-type IRS for isoleucine, dl-valine, thiaisoleucine, and O-methyl-dl-threonine, both in the pyrophosphate exchange assay and in the assay of isoleucyl-tRNA formation. The affinity of the mutant enzyme for adenosine triphosphate in the assay of isoleucyl-tRNA formation is 15-fold lower than that of the wild-type enzyme. The affinity of mutant IRS for tRNA is not changed as compared with wild-type IRS. These data show that mutant IRS has an altered structure and clearly confirm that ileS is the structural gene for IRS.
D000255:Adenosine Triphosphate / Q000378:metabolism; D002247:Carbon Isotopes; D003470:Culture Media; D011756:Diphosphates / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D005796:Genes; D005827:Genetics, Microbial; D006358:Hot Temperature; D007532:Isoleucine / Q000378:metabolism; D008025:Ligases / Q000302:isolation & purification / Q000378:metabolism*; D009154:Mutation; D010759:Phosphorus Isotopes; D012343:RNA, Transfer / Q000378:metabolism; D013997:Time Factors; D014633:Valine
D002247:Carbon Isotopes; D003470:Culture Media; D011756:Diphosphates; D010759:Phosphorus Isotopes; D007532:Isoleucine; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D008025:Ligases; D014633:Valine