Synthesis of homocysteine thiolactone by methionyl‐tRNA synthetase in cultured mammalian cells

Abstract

Homocysteine thiolactone is a product of an error-editing reaction, catalyzed by Escherrchiu colr and Saccharomyces cerevrsiae methionyl-tRNA synthetases, which prevents incorporation of homocysteine into tRNA and protein both in vitro and in vivo. Here, homocysteine thiolactone is also shown to be synthesized by cultured mammalian cells such as human cervical carcinoma (HeLa), mouse renal adenocarcinoma (RAG), and Chinese hamster ovary (CHO) cells labeled with ['5S]methionine, but not by normal human and mouse (Balblc 3T3) fibroblasts. A temperaturesensitive methionyl-tRNA synthetase mutant of CHO cells, Met-l, does not make the thiolactone at the non-permissive temperature. The data indicate that methionyl-tRNA synthetase is involved in synthesis of homocysteine thiolactone in CHO cells, thereby extending this important proofreading mechanism to mammalian cells.

Editing function ofEscherichia coli cysteinyl-tRNA synthetase: cyclization of cysteine to cysteine thiolactone

“…Similar ratios of deacylating and synthetic activities involving cognate substrates have been observed with other tRNAs and synthetases (10,18,19). Because of a relative slowness of enzymatic deacylation, in each case other explanations for the origin of the observed deacyaltion of aminoacyl-tRNA, in particular a possibility of a contaminating enzyme, must be ruled out.…”

“…Homocysteine is misactivated by methionyl-tRNA synthetase at unacceptably high level (4-6) but then cyclized to homocysteine thiolactone (7) to prevent transfer to tRNAMet both in vitro (5,7) and in vivo (8)(9)(10). Because of limited selectivity of the editing site of methionyl-tRNA synthetase, some of the cognate substrate methionine is also cyclized to a corresponding thiolactone, S-methyl homocysteine thiolactone (1 1).…”

A novel pathway for the conversion of homocysteine to methionine in eukaryotes

“…Our data suggests that the methylation reaction is essential and we propose that, in the absence of methylation, Hcy-tRNA is hydrolysed to produce homocysteine thiolactone. Notably, previous studies have shown that homocysteine thiolactone formation is especially high in cells defective in methionine metabolism [3].…”

“…Hydrolysis of homocysteinyl-AMP gave rise to homocysteine thiolactone and AMP [2]. Based on this and other experiments in i o, it was postulated that the formation of large quantities of homocysteine thiolactone by several types of malignant cells is due to the proofreading activity of Met-tRNA synthetase [3]. Misactivation was measured by measuring the thiolactone formed.…”

“…In the absence of methylation, tRNA-attached homocysteine is hydrolysed to produce homocysteine thiolactone. sible for homocysteine thiolactone formation, the extent of homocysteine misactivation reported is extremely high both in i o and in itro [2,3,5]. One would, therefore, expect a small fraction of the misactivated homocysteine to escape the proofreading activity of the synthetase and appear as Hcy-tRNA.…”