The proofreading of hydroxy analogues of leucine and isoleucine by leucyl-tRNA synthetases from E. coli and yeast.
Three analogues each of leucine and isoleucine carrying hydroxy groups in gamma- or delta- or gamma- and delta-position have been synthesized, and tested in the aminoacylation by leucyl-tRNA synthetases from E. coli and yeast. Hydrolytic proofreading, as proposed in the chemical proofreading model, of these analogues and of homocysteine should result in a lactonisation of these compounds and therefore provide information regarding the proofreading mechanism of the two leucyl-tRNA synthetases. Leucyl-tRNA synthetase from E. coli shows a high initial substrate discrimination. Only two analogues, gamma-hydroxyleucine and homocysteine are activated and transferred to tRNALeu where a post-transfer proofreading occurs. Lactonisation of gamma-hydroxyleucine and homocysteine could be detected. Leucyl-tRNA synthetase from yeast has a relatively poor initial discrimination of these substrates, which is compensated by a very effective pre-transfer proofreading on the aminoacyl-adenylate level. No lactonisation nor mischarged tRNALeu is detectable.
D000249:Adenosine Monophosphate / Q000096:biosynthesis; D000604:Amino Acyl-tRNA Synthetases / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology; D006710:Homocysteine / Q000378:metabolism; D006868:Hydrolysis; D007532:Isoleucine / Q000378:metabolism*; D007783:Lactones / Q000378:metabolism; D007930:Leucine / Q000378:metabolism*; D007935:Leucine-tRNA Ligase / Q000502:physiology*; D014176:Protein Biosynthesis; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D007783:Lactones; D012346:RNA, Transfer, Amino Acyl; D007532:Isoleucine; D006710:Homocysteine; D000249:Adenosine Monophosphate; D000604:Amino Acyl-tRNA Synthetases; D007935:Leucine-tRNA Ligase; D007930:Leucine