Search

High-fat diet is associated with elevated plasma homocysteine (Hcy), and both are linked to cancer. Although Hcy is not a coded amino acid, proteins do carry Hcy modifications formed via a pathway involving methionyl-tRNA synthetase-catalyzed metabolic conversion of Hcy to Hcy-thiolactone. Hcy-thiolactone then chemically reacts with protein lysine residues, affording KHcy-protein. Recently, Wang et al.[1](Cell Rep. 2018;25:398–412.e6) showed that this pathway promotes colorectal cancer by impairing DNA damage repair.

The universally conserved translation elongation factor EF-Tu delivers aminoacyl(aa)-tRNA in the form of an aa-tRNA·EF-Tu·GTP ternary complex (TC) to the ribosome where it binds to the cognate mRNA codon within the ribosomal A-site, leading to formation of a pretranslocation (PRE) complex. Here we describe preparation of QSY9 and Cy5 derivatives of the variant E348C-EF-Tu that are functional in translation elongation. Together with fluorophore derivatives of aa-tRNA and of ribosomal protein L11, located within the GTPase associated center (GAC), these labeled EF-Tus allow development of two new FRET assays that permit the dynamics of distance changes between EF-Tu and both L11 (Tu-L11 assay) and aa-tRNA (Tu-tRNA assay) to be determined during the decoding process. We use these assays to examine: (i) the relative rates of EF-Tu movement away from the GAC and from aa-tRNA during decoding, (ii) the effects of the misreading-inducing antibiotics streptomycin and paromomycin on tRNA selection at the A-site, and (iii) how strengthening the binding of aa-tRNA to EF-Tu affects the rate of EF-Tu movement away from L11 on the ribosome. These FRET assays have the potential to be adapted for high throughput screening of ribosomal antibiotics.

Faithful translation of the genetic code during protein synthesis is fundamental to the growth, development, and function of living organisms. Aminoacyl‐tRNA synthetases (AARSs), which define the genetic code by correctly pairing amino acids with their cognate tRNAs, are responsible for ‘quality control’ in the flow of information from a gene to a protein. When differences in binding energies of amino acids to an AARS are inadequate, editing is used to achieve high selectivity. Editing occurs at the synthetic active site by hydrolysis of noncognate aminoacyl‐adenylates (pretransfer editing) and at a dedicated editing site located in a separate domain by deacylation of mischarged aminoacyl‐tRNA (posttransfer editing). Access of nonprotein amino acids, such as homocysteine or ornithine, to the genetic code is prevented by the editing function of AARSs, which functionally partitions amino acids present in living cells into proteinand nonproteinamino acids. Continuous editing is part of the tRNA aminoacylation process in living organisms from bacteria to human beings. Preventing mistranslation by the clearance of misactivated amino acids is crucial to cellular homeostasis and has a role in etiology of disease. Although there is a strong selective pressure to minimize mistranslation, some organisms possess error‐prone AARSs that cause mistranslation. Elevated levels of mistranslation and the synthesis of statistical proteins can be beneficial for pathogens by increasing phenotypic variation essential for the evasion of host defenses. WIREs RNA2012, 3:295–310. doi: 10.1002/wrna.122

Chemical reactivity of homocysteine thiolactone (HTL) has been implicated in cardiovascular disease. Owing to its aminoacyl‐thioester character, HTL undergoes facile electrophilic and nucleophilic reactions at its amino and activated‐carboxyl group, respectively. To gain insight into the mechanism of the reactions involving its amino group, the kinetics of the condensation of homocysteine thiolactone with formaldehyde, acetaldehyde, and pyridoxal phosphate, were analyzed in the pH range from 5 to 10. The reactions were first order with respect to HTL, aldehyde, and hydroxide ion concentrations. Of the two ionic species of HTL (pKa=6.67±0.05), the acid form HTL+was ∼100‐fold more reactive than the base form HTL0. The reactions of HTL with aldehydes involve intermediate adducts. The conversion of the intermediate carbinolamine to a product, 1,3‐tetrahydrothiazine‐4‐carboxylic acid or its 2‐substituted analogue, occurs in a two‐step reaction. The first step involves hydrolysis of the thioester bond in the intermediate, facilitated by anchimeric assistance by the oxygen of the carbinolamine group of the intermediate. The second step involves an attack of the liberated thiolate on the aldehyde‐derived carbon of the intermediate, affording 1,3‐tetrahydrothiazine‐4‐carboxylic acid or its 2‐substituted analogue. An unusual feature of these reactions is that the formation of the carbinolamine group increases the reactivity of the thioester bond of HTL ∼104‐fold. The facile formation of tetrahydrothiazines may contribute to HTL elimination from the human body.

Homocysteine is thought to be a non-protein amino acid. However, in vitro studies suggest that homocysteine is likely to be incorporated by indirect mechanisms into proteins in living organisms. Here I show that homocysteine is a protein amino acid in humans. Homocysteine bound by amide or peptide linkages (Hcy-N-protein) is present in human hemoglobin, serum albumin, and gamma-globulins. 1 molecule of homocysteine per 1000 or 1670 molecules of methionine was present in hemoglobin or albumin, respectively. Other proteins, such as low density lipoprotein, high density lipoprotein, transferrin, antitrypsin, and fibrinogen, contained lower amounts of Hcy-N-protein. In human plasma, levels of Hcy-N-protein represented from 0.3 to 23% of total homocysteine. Thus, Hcy-N-protein is a significant component of homocysteine metabolism in humans, possibly contributing to adverse effects of homocysteine on human cells.

The yeast Saccharomyces cerevisiae possesses two methionyl-tRNA synthetases (MetRS), one in the cytoplasm and the other in mitochondria. The cytoplasmic MetRS has a zinc-finger motif of the type Cys-X2-Cys-X9-Cys-X2-Cys in an insertion domain that divides the nucleotide-binding fold into two halves, whereas no such motif is present in the mitochondrial MetRS. Here, we show that tightly bound zinc atom is present in the cytoplasmic MetRS but not in the mitochondrial MetRS. To test whether the presence of a zinc-binding site is required for cytoplasmic functions of MetRS, we constructed a yeast strain in which cytoplasmic MetRS gene was inactivated and the mitochondrial MetRS gene was expressed in the cytoplasm. Provided that methionine-accepting tRNA is overexpressed, this strain was viable, indicating that mitochondrial MetRS was able to aminoacylate tRNAMet in the cytoplasm. Site-directed mutagenesis demonstrated that the zinc domain was required for the stability and consequently for the activity of cytoplasmic MetRS. Mitochondrial MetRS, like cytoplasmic MetRS, supported homocysteine editing in vivo in the yeast cytoplasm. Both MetRSs catalyzed homocysteine editing and aminoacylation of coenzyme A in vitro. Thus, identical synthetic and editing functions can be carried out in different structural frameworks of cytoplasmic and mitochondrial MetRSs.

A metabolite of homocysteine (Hcy), the thioester Hcy thiolactone, damages proteins by modifying their lysine residues which may underlie Hcy‐associated cardiovascular disease in humans. A protein component of high density lipoprotein, Hcy thiolactonase (HTase) hydrolyzes thiolactone to Hcy. Thiolactonase is a product of the polymorphic PON1gene, also involved in detoxification of organophospates and implicated in cardiovascular disease. Polymorphism in PON1affects the detoxifying activity of PON1 in a substrate‐dependent manner. However, how PON1polymorphism affects HTase activity is unknown. Here we report a strong association between the thiolactonase activity and PON1genotype in human populations. High thiolactonase activity was associated with L55 and R192 alleles, more frequent in blacks than in whites. Low thiolactonase activity was associated with M55 and Q192 alleles, more frequent in whites than in blacks. High thiolactonase activity afforded better protection against protein homocysteinylation than low thiolactonase activity. These results suggest that variations in HTase may play a role in Hcy‐associated cardiovascular disease.

Coenzyme A (CoA-SH), a cofactor in carboxyl group activation reactions, carries out a function in nonribosomal peptide synthesis that is analogous to the function of tRNA in ribosomal protein synthesis. The amino acid selectivity in the synthesis of aminoacyl-thioesters by nonribosomal peptide synthetases is relaxed, whereas the amino acid selectivity in the synthesis of aminoacyl-tRNA by aminoacyl-tRNA synthetases is restricted. Here I show that isoleucyl-tRNA synthetase aminoacylates CoA-SH with valine, leucine, threonine, alanine, and serine in addition to isoleucine. Valyl-tRNA synthetase catalyzes aminoacylations of CoA-SH with valine, threonine, alanine, serine, and isoleucine. Lysyl-tRNA synthetase aminoacylates CoA-SH with lysine, leucine, threonine, alanine, valine, and isoleucine. Thus, isoleucyl-, valyl-, and lysyl-tRNA synthetases behave as aminoacyl-S-CoA synthetases with relaxed amino acid selectivity. In contrast, RNA minihelices comprised of the acceptor-TψC helix of tRNAIleor tRNAValwere aminoacylated by cognate synthetases selectively with isoleucine or valine, respectively. These and other data support a hypothesis that the present day aminoacyl-tRNA synthetases originated from ancestral forms that were involved in noncoded thioester-dependent peptide synthesis, functionally similar to the present day nonribosomal peptide synthetases.

The non-protein amino acid homocysteine (Hcy), owing to its structural similarity to the protein amino acids methionine, isoleucine, and leucine, enters first steps of protein synthesis and is activated by methionyl-, isoleucyl-, and leucyl-tRNA synthetases in vivo. However, translational incorporation of Hcy into protein is prevented by editing mechanisms of these synthetases, which convert misactivated Hcy into thiolactone. The lack of efficient interactions of the side chain of Hcy with the specificity subsite of the synthetic/editing active site is a prerequisite for editing of Hcy. Thus, if the side chain thiol of Hcy were reversibly modified with a small molecule that would enhance its binding to the specificity subsite and prevent editing, such modified Hcy is predicted to be transferred to tRNA and incorporated translationally into protein. Here I show thatS-nitroso-Hcy is in fact transferred to tRNA by methionyl-tRNA synthetase and incorporated into protein by the bacterium Escherichia coli. S-Nitroso-Hcy-tRNA also supports translation of mRNAs in a rabbit reticulocyte system. Removal of the nitroso group yields Hcy-tRNA and protein containing Hcy in peptide bonds. S-Nitrosylation-mediated translational incorporation of Hcy into protein may occur under natural conditions in cells and contribute to Hcy-induced pathogenesis in atherosclerosis.

Homocysteine thiolactone is formed in all cell types studied thus far as a result of editing reactions of some aminoacyl-tRNA synthetases. Because inadvertent reactions of thiolactone with proteins are potentially harmful, the ability to detoxify homocysteine thiolactone is essential for biological integrity. This work shows that a single specific enzyme, present in mammalian but not in avian sera, hydrolyzes thiolactone to homocysteine. Human serum thiolactonase, a 45-kDa protein component of high density lipoprotein, requires calcium for activity and stability and is inhibited by isoleucine and penicillamine. Substrate specificity studies suggest that homocysteine thiolactone is a likely natural substrate of this enzyme. However, thiolactonase also hydrolyzes non-natural substrates, such as phenyl acetate,p-nitrophenyl acetate, and the organophospate paraoxon. N-terminal amino acid sequence of pure thiolactonase is identical with that of human paraoxonase. These and other data indicate that paraoxonase, an organophosphate-detoxifying enzyme whose natural substrate and function remained unknown up to now, is in fact homocysteine thiolactonase. By detoxifying homocysteine thiolactone, the thiolactonase/paraoxonase would protect proteins against homocysteinylation, a potential contributing factor to atherosclerosis.

Homocysteine thiolactone, a cyclic thioester, is synthesized by certain aminoacyl‐tRNA synthetases in editing or proofreading reactions that prevent translational incorporation of homocysteine into proteins. Although homocysteine thiolactone is expected to acylate amino groups in proteins, virtually nothing is known regarding reactivity of the thiolactone. Here it is shown that reactions of the thiolactone with protein lysine residues were robust under physiological conditions. In human serum incubated with homocysteine thiolactone, protein homocysteinylation was a major reaction that could be observed with as little as 10 nM thiolactone. Individual proteins were homocysteinylated at rates proportional to their lysine contents. Homocysteinylation led to protein damage, manifested as multimerization and precipitation of extensively modified proteins. Model enzymes, such as methionyl‐tRNA synthetase and trypsin, were inactivated by homocysteinylation. Metabolic conversion of homocysteine to the thiolactone, protein homocysteinylation, and resulting protein damage may underlie involvement of Hcy in the pathology of vascular disease.—Jakubowski, H. Protein homocysteinylation: possible mechanism underlying pathological consequences of elevated homocysteine levels. FASEB J.13, 2277–2283 (1999)

Previous in vitro work has shown that Escherichia colimethionyl‐tRNA synthetase has a limited ability to discriminate against cognate methionine in the editing site designed for noncognate homocysteine. As a result, a small fraction of the correct product Met‐tRNA is deacylated with the formation of a cyclic sulfonium compound, S‐methyl‐homocysteine thiolactone. This is exploited here to estimate energy costs associated with the destruction of a correct product by methionyl‐tRNA synthetase in bacterial cells. In vivo measurements of S‐methyl‐homocysteine thiolactone indicate that in Escherichia coli 3.3molecules of Met‐tRNA are destroyed by deacylation per 10,000 molecules of Met‐tRNA successfully transferring methionine to protein. This number of destroyed molecules of a correct product, Met‐tRNA, is 30 times lower than the number of destroyed molecules of an incorrect product, homocysteinyl adenylate. Thus, most of the energy cost of proofreading in vivo is due to editing of the noncognate amino acid.— Jakubowski, H. Energy cost of proofreading in vivo: the charging of methionine tRNAs in Escherichia coli. FASEB J.7: 168‐172; 1993.

Editing of the non-protein amino acid homocysteine, a frequent type of error-correcting process in amino acid selection for protein synthesis by an aminoacyl-tRNA synthetase, results in formation of a cyclic thioester, homocysteine thiolactone. Here it is shown that human cells in which homocysteine metabolism is deregulated by a mutation in the cystathionine β-synthase gene and/or by an antifolate drug, aminopterin (which prevents remethylation of homocysteine to methionine by methionine synthase), produce more homocysteine thiolactone, in addition to homocysteine, than unaffected cells. The thiolactone is incorporated into cellular and extracellular proteins, in addition to being secreted and hydrolyzed to homocysteine. Experiments with model proteins and amino acids suggest that the mechanism of incorporation involves acylation of side chain amino groups of lysine residues by the activated carboxyl group of the thiolactone. The metabolic conversion of homocysteine to homocysteine thiolactone and the reactivity of the thiolactone toward proteins may explain pathological consequences of elevated levels of homocysteine such as observed in vascular disease.

Homoeysteine thiolactone is a product of an error-editing reaction, catalyzed by Escherichia coliand Saccharomyces cerevisiaemethionyl-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 [ 35S]methionine, but not by normal human and mouse (Balb/c 3T3) fibroblasts. A temperature-sensitive methionyl-tRNA synthetase mutant of CHO cells, Met-1, 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.

In the modified release factor 2 (RF2) programmed translational frameshift (with a sense codon replacing the wild-type in-frame UGA codon at the shift site), ribosomes shift+1 into the reading frame for an out-of-frame reporter fused to the frameshift sequence. Partitioning of ribosomes between the out-of-frame shift and in-frame reading depends on the codon at the shift site and on the levels of tRNA decoding the in-frame codon. Overexpression of a tRNA species cognate to the in-frame codon at the shift site significantly reduces the frequency of frame-shifting, presumably by facilitating in-frame reading, which would reduce production of the out-of-frame reporter. However, since overexpression of a tRNA increases levels of both charged and uncharged tRNA, it is possible that uncharged cognate tRNA might be able to reduce the frequency of the frameshift, by entering the A site on the ribosome. To test this, we manipulated charged and uncharged tRNA levelsin vivo, using the tryptophan analog tryptophan hydroxamate, which increases the proportion of uncharged tRNA^Trpby competing with cognate amino acid tryptophan for tryptophanyl-tRNA synthetase, thereby reducing protein synthesis. We report here that a slight but reproducible reduction in the relative frequency of the frameshift is observed when tryptophan hydroxamate is added to cells containing the modified RF2 shift with UGG (Trp codon) at the shift site. When tRNA^Trpis overexpressed from another plasmid, the shift frequency drops three- to fourfold, as expected, however, this reduction is still seen in the presence of the analog. Thus, under conditions when most of the tRNA^Trpis apparently uncharged, excess tRNA^Trpstill causes a significant reduction in the frameshift when UGG is at the shift site, providing evidence that uncharged cognate tRNA also can inhibit this frameshift.

Homoeysteine thiolactone is a product of an error‐editing reaction, catalyzed by Escherichia coliand Saccharomyces cerevisiaemethionyl‐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 [35S]methionine, but not by normal human and mouse (Balb/c 3T3) fibroblasts. A temperature‐sensitive methionyl‐tRNA synthetase mutant of CHO cells, Met‐1, 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.

Protein N-homocysteinylation by a homocysteine (Hcy) metabolite, Hcy-thiolactone, is an emerging post-translational modification (PTM) that occurs in all tested organisms and has been linked to human diseases. The yeast Saccharomyces cerevisiaeis widely used as a model eukaryotic organism in biomedical research, including studies of protein PTMs. However, patterns of global protein N-homocysteinylation in yeast are not known. Here, we identified 68 in vivoand 197 in vitro N-homocysteinylation sites at protein lysine residues (N-Hcy-Lys). Some of the N-homocysteinylation sites overlap with other previously identified PTM sites. Protein N-homocysteinylation in vivo, induced by supplementation of yeast cultures with Hcy, which elevates Hcy-thiolactone levels, was accompanied by significant changes in the levels of 70 yeast proteins (38 up-regulated and 32 down-regulated) involved in the ribosomal structure, amino acid biosynthesis, and basic cellular pathways. Our study provides the first global survey of N-homocysteinylation and accompanying changes in the yeast proteome caused by elevated Hcy level. These findings suggest that protein N-homocysteinylation and dysregulation of cellular proteostasis may contribute to the toxicity of Hcy in yeast. Homologous proteins and N-homocysteinylation sites are likely to be involved in Hcy-related pathophysiology in humans and experimental animals. Data are available via ProteomeXchange with identifier PXD020821.

Abundant corneocyte surface protrusions, observed in patients with atopic dermatitis with filaggrin loss-of-function mutations, are inversely associated with levels of natural moisturizing factors (NMFs) in the stratum corneum. To dissect the etiological role of NMFs and filaggrin deficiency in surface texture alterations, we examined mouse models with genetic deficiencies in the synthesis or degradation of filaggrin monomers for NMFs, cell stiffness (elastic modulus) and corneocyte surface protrusion density (dermal texture index). Five neonatal and adult mouse models carrying inactivating mutations of SASPase (Sasp−/−), filaggrin (Flgft/ftand Flg−/−), filaggrin-hornerin (FlgHrnr−/−), and bleomycin hydrolase (Blmh−/−) were investigated. Sasp−/−and Flg−/−were on the hairless mouse background. Atomic force microscopy was used to determine elastic modulus and dermal texture index. Corneocytes of each neonatal as well as hairless adult knockout mouse exhibited an increased number of protrusions and decreased elastic modulus. In these mice, NMFs were reduced except for Sasp−/−. Dermal texture index was inversely correlated with NMFs and elastic modulus. Our findings demonstrate that any filaggrin-NMF axis deficiency can affect corneocyte mechanical properties in mice and likely in humans. Differences in NMFs and corneocyte surface texture between neonatal and adult as well as hairless and hairy mice emphasize the need for carefully selecting the most appropriate animal models for studies.

Increased levels of plasma homocysteine is an independent risk factor for cardiovascular disease and has cell-type distinct proatherosclerotic effects on vascular cells. In this study, we characterized L- homocysteine transport in cultured human aortic endothelial and aortic smooth muscle cells. L-homocysteine was transported into vascular cells in a time-dependent fashion. L-homocysteine transport activity was about 2-fold higher in aortic smooth muscle cells. In addition, L-homocysteine transport in both cell types was mediated by sodium-dependent and independent carrier systems. Competition studies revealed that the neutral amino acids cysteine, glycine, serine, tyrosine, alanine, leucine, and methionine, and inhibitors of the cysteine transport systems inhibited L-homocysteine uptake in both cell types, but the inhibition was greater in endothelial cells. Eadie-Hofstee plots demonstrated that L-Hcy transport in endothelial cells had a Michaelis constant (Km) of 79mM and a maximum transport velocity (Vmax) of 873 pmol/mg protein/min. In contrast, homocysteine transport in aortic smooth muscle cells had a lower affinity (Km=212mM) but a higher transport capacity (Vmax=4192 pmol/mg protein/min). Interestingly, increases in pH (pH 6.5–8.2) only inhibited L-homocysteine uptake in endothelial cells. Moreover, L-homocysteine transport in endothelial cells was partially inhibited by lysosomal inhibitors. Our studies indicate that L-homocysteine shares transporter systems with cysteine and can be inhibited for transport by multiple neutral amino acids in vascular cells, and that L-homocysteine transport involves lysosomal transport in endothelial cells. The specific lysosomic feature of L-homocystein transport in endothelial cells may contribute to cell type specific growth inhibitory effects and therefore play a role in homocysteine atherogenic potential.

Increased levels of plasma homocysteine is an independent risk factor for cardiovascular disease and has cell-type distinct proatherosclerotic effects on vascular cells. In this study, we characterized L- homocysteine transport in cultured human aortic endothelial and aortic smooth muscle cells. L-homocysteine was transported into vascular cells in a time-dependent fashion. L-homocysteine transport activity was about 2-fold higher in aortic smooth muscle cells. In addition, L-homocysteine transport in both cell types was mediated by sodium-dependent and independent carrier systems. Competition studies revealed that the neutral amino acids cysteine, glycine, serine, tyrosine, alanine, leucine, and methionine, and inhibitors of the cysteine transport systems inhibited L-homocysteine uptake in both cell types, but the inhibition was greater in endothelial cells. Eadie-Hofstee plots demonstrated that L-Hcy transport in endothelial cells had a Michaelis constant (Km)of 79mM and a maximum transport velocity (Vmax)of 873 pmol/mg protein/min. In contrast, homocysteine transport in aortic smooth muscle cells had a lower affinity (Km=212mM) but a higher transport capacity (Vmax=4192 pmol/mg protein/min). Interestingly, increases in pH (pH 6.5–8.2) only inhibited L-homocysteine uptake in endothelial cells. Moreover, L-homocysteine transport in endothelial cells was partially inhibited by lysosomal inhibitors. Our studies indicate that L-homocysteine shares transporter systems with cysteine and can be inhibited for transport by multiple neutral amino acids in vascular cells, and that L-homocysteine transport involves lysosomal transport in endothelial cells. The specific lysosomic feature of L-homocystein transport in endothelial cells may contribute to cell type specific growth inhibitory effects and therefore play a role in homocysteine atherogenic potential.

Evidence is presented that the editing mechanisms of aminoacyl-tRNA synthetases operate by two alternative pathways: pre-transfer, by hydrolysis of the non-cognate aminoacyl adenylate; post-transfer, by hydrolysis of the mischarged tRNA. The methionyl-tRNA synthetases from Escherichia coli and Bacillus stearothermophilus and isoleucyl-tRNA synthetase from E. coli, for example, are shown to reject misactivated homocysteine rapidly by the pretransfer route. A novel feature of this reaction is that homocysteine thiolactone is formed by the facile cyclisation of the homocysteinyl adenylate. Valyl-tRNA synthetases, on the other hand, reject the more readily activated non-cognate amino acids by primarily the post-transfer route. The features governing the choice of pathway are discussed.

Editing of errors in amino acid selection by an aminoacyl-tRNA synthetase prevents attachment of incorrect amino acids to tRNA, thereby greatly enhancing accuracy of translation of the genetic code. Editing of the non-protein amino acid homocysteine, a frequent type of an error-correcting process, involves reaction of the side chain sulfhydryl group of homocysteine with its activated carboxyl group forming a cyclic thioester, homocysteine thiolactone. Here, it is shown that isoleucyl-tRNA synthetase (lleRS), which occasionally misactivates homocysteine in vitro and in vivo, catalyzes reactions of activated isoleucine with organic thiols (analogues of the side chain of homocysteine). That these enzymatic reactions occur between Ile-tRNAIle or Ile-AMP (bound in the synthetic sub-site) and a thiol (an analogue of the side chain of homocysteine, bound in the editing sub-site), indicates that the two sub-sites are physically close on the surface of IleRS, forming a single synthetic/editing active site of the enzyme. Although IleRS•Val-AMP undergoes thiolysis as efficiently as do IleRS•Ile-AMP and IleRS•Ile-tRNAIle, IleRS•Val-tRNAIle does not react with thiols. These and other data suggest that the mischarged valine residue in IleRS•Val-tRNAIle is, most likely, positioned off the enzyme.

A cyclic sulfur compound, identified as cysteine thiolactone by several chemical and enzymatic tests, is formed from cysteine during in vitro tRNACys aminoacylation catalyzed by Escherichia coli cystelnyl-tRNA synthetase. The mechanism of cysteine thiolactone formation involves enzymatic deacylatlon of Cys-tRNACys (k = 0.017 s−1) in which nucleophlllc sulfur of the side chain of cysteine in Cys-tRNACys attacks its carboxyl carbon to yield cysteine thiolactone. Nonenzymatic deacylatlon of Cys-tRNACys (k = 0.0006 s−1) yields cysteine, as expected. Inhibition of enzymatic deacylation of Cys-tRNACys by cysteine and Cys-AMP, but not by ATP, indicates that both synthesis of Cys-tRNACys and cyclization of cysteine to the thiolactone occur in a single active site of the enzyme. The cyclization of cysteine is mechanistically similar to the editing reactions of methlonyl-tRNA synthetase. However, in contrast to methionyl-tRNA synthetase which needs the editing function to reject misactivated homocysteine, cysteinyl-tRNA synthetase is highly selective and is not faced with a problem in rejecting noncognate amlno acids. Despite this, the present day cysteinyl-tRNA synthetase, like methionyl-tRNA synthetase, still retains an editing activity toward the cognate product, the charged tRNA. This function may be a remnant of a chemistry used by an ancestral cysteinyl-tRNA synthetase.

Arglnyl-tRNA synthetase (ArgRS) catalyses AMP- and PP1independent deacylation of Arg-tRNAArg9 in the presence of cysteine. A dipeptide, Arg-Cys, is a product of this deacylation reaction. Similar reaction with homocysteine yields Arg-Hcy. Arginine is a noncompetitive inhibitor of the cysteine-dependent deacylation which indicates that cysteine binds to the enzyme-Arg-tRNAArg.9 complex at a site separate from the arginine binding site. In the presence of arginine, [14C]Arg-tRNAArg is deacylated at a rate similar to the rate of its spontaneous deacylation in solution and [14C]arginine is a product. Experiments with cysteine derivatives indicate that the -SH group is essential for the reaction whereas -NH2 and -COOH groups are not. Thloesters of arginine are formed with 3-mercaptopropionic acid, N-acetyl-L-cysteine and dithiothreitol. These data suggest that formation of the dipeptide Arg-Cys involves a thioester intermediate, S->L-arginyl)- L-cysteine, which is not observed because of the rapid rearrangement to form a stable peptide bond. Facile intramolecular reaction results from the favorable geometric arrangement of the a-amino group of cysteine with respect to the thioester formed in the initial reaction. Similar reactions, yielding Ile-Cys and Val-Cys, are catalyzed by isoleucyl- and valyl-tRNA synthetases, respectively.