Aminoacyl cysteines

To prevent the S—>N acyl shift, S-palmitoylation with palmitoyl chloride is performed in anhydrous solvents on N -aminoacylated cysteines, e.g. dipeptide building blocks, whereby the residual functionalities of the peptide have to be protected taking into account the base-lability of the thioester bond. 

Examination of the complete genome sequences of methanogens revealed an apparent lack of cysteinyl-tRNA synthetase. However, prolyl-tRNA synthetase does correctly aminoacylate the tRNAs for both proline and cysteine in these archaeobacteria.271 272a... 

The synthetase consists of the three modules E1, E2, and E3 (for a complete description, see Sec. II. A). Each module is composed of an activation site forming the acyl or aminoacyl adenylate, a carrier domain which is posttranslationally modified with 4 -phosphopantetheine (Sp), and a condensation domain (Cl, C2) or, alternatively, a structurally similar epimerization domain (Ep). Activation of aminoadipate (Aad) leads to an acylated enzyme intermediate, in which Aad is attached to the terminal cysteamine of the cofactor (El-Spl-Aad) [reactions (1) and (2)]. Likewise, activation of cysteine (Cys) leads to cysteinylated module 2 [reactions (3) and (4)]. For the condensation reaction to occur between aminoadipate as donor and cysteine as acceptor, both intermediates are thought to react at the condensation site of module 1 (Cl). Each condensation site is composed, in analogy to ribosomal peptide formation, of an aminoacyl and a peptidyl site. In this case of initiation, the thioester of Aad enters the P-site, while the thioester of Cys enters the A-site. Condensation occurs and leaves the dipeptidyl intermediate Aad-Cys at the carrier protein of the second module [reaction (5)]. The third amino acid valine is activated on module 3, and Val is attached to the carrier protein 3 [reactions (6) and (7)]. Formation of the tripeptide occurs at the second condensation site C2, with the dipeptidyl intermediate entering the P-site and the valiny 1-intermediate the A-site [reaction (8)]. 

Comprehensive Biological Catalysis—a Mechanistic Reference Volume has recently been published. The fiiU contents list (approximate number of references in parentheses) is as follows S-adenosylmethionine-dependent methyltransferases (110) prenyl transfer and the enzymes of terpenoid and steroid biosynthesis (330) glycosyl transfer (800) mechanism of folate-requiring enzymes in one-carbon metabohsm (260) hydride and alkyl group shifts in the reactions of aldehydes and ketones (150) phosphoenolpyruvate as an electrophile carboxyvinyl transfer reactions (140) physical organic chemistry of acyl transfer reactions (220) catalytic mechanisms of the aspartic proteinases (90) the serine proteinases (135) cysteine proteinases (350) zinc proteinases (200) esterases and lipases (160) reactions of carbon at the carbon dioxide level of oxidation (390) transfer of the POj group (230) phosphate diesterases and triesterases (160) ribozymes (70) catalysis of tRNA aminoacylation by class I and class II aminoacyl-tRNA synthetases (220) thio-disulfide exchange of divalent sulfirr (150) and sulfotransferases (50). 

A new translation. A transfer RNA with a UGU anticodon is enzymatically conjugated to i C-labeled cysteine. The cysteine unit is then chemically modified to alanine (with the use of Raney nickel, which removes the sulfur atom of cysteine). The altered aminoacyl-tRNA is added to a protein-synthesizing system containing normal components except for this tRNA. The mRNA added to this mixture contains the following sequence ... 

Does this mischarged tRNA recognize the codon for cysteine or for alanine The answer came when the tRNA was added to a cell-free protein-synthesizing system. The template was a random copolymer of U and G in the ratio of 5 1, which normally incorporates cysteine (encoded by UGU) but not alanine (encoded by GCN). However, alanine was incorporated into a polypeptide when Ala-tHNA ys was added to the incubation mixture. The same result was obtained when mRNA for hemoglobin served as the template and [i CJalanyl-tRNACys was used as the mischarged aminoacyl-tRNA. The only radioactive tryptic peptide produced was one that normally contained cysteine but not alanine. Thus, the amino acid in aminoacyl-tRNA does not play a role in selecting a codon. 

Kamtekar S, Kennedy WD, Wang J, Stathopoulos C, Soil D, Steitz TA The structural basis of cysteine aminoacylation of tRNAPro by prolyl-tRNA synthetases. Proc Natl Acad Sci USA 2003, 100(4) 1673—1678. 

Amino acid metabolism A artate aminotransferase Alanine aminotransferase Cysteine aminotransferase Tyrosine aminotransferase Leucine aminotransferase Alanine-ketoacid aminotransfoase Ornithine-ketoacid aminotransferase A artate carbamoyl transferase Methionine adenosyl transferase Glutamate decarboxylase Glutamate dehydrogenase Serine hydroxymethyltransferase Aminoacyl-sRNA synthetases... 

Aminoacylation has initially been ascribed to cysieinyl side chains (13), until peptide synthetase sequencing work first completed on the ACV synthetase from PeniciUium cfirysogenurn revealed the absence of conserved cysteine residues. With a considerable... 

This was demonstrated by taking the aminoacyl-tRNA complex specific for cysteine (abbreviation, cysteinyl-tRNA showing that the cysteine has combined with its specific tRNA) and modifying the amino acid side chain to form alanine by catalytic reduction. 

The modified aminoacyl-tRNA was then incubated in vivo and incorporated alanine into the protein at those positions which normally accepted cysteine. 

According to the mechanism of translation outlined in the introduction, it is clear that analogues are activated before being incorporated into protein, and compete with the protein amino acid for the aminoacyl-transfer-RNA synthetases. They are afterwards transferred to specific transfer RNAs. When the natural amino-acid analogue has been transferred to the transfer-RNA molecule, it takes no part in determining the specificity of polypeptide synthesis. The incorporation of Ala in place of Cys, after the chemical reduction of Cys-tRNA has been outlined before (see Para. 8.1.2). Similarly Cys-tRNA can be mildly oxidized to CysSOsH-tRNA thereafter cysteine sulfonic acid becomes incorporated into protein. This appears to be a suitable procedure to incorporate an analogue which per se is not specific enough to be incorporated (CysSOsH itself is not incorporated). Such a procedure is, however, essentially restricted to in vitro studies. 

This conformation is both able to bind aminoacylated tRNA and bind to the ribosome. Unfortunately the fragmented protein binds neither GTP nor tRNA. However, it is known that the aminoacyl end of the tRNA interacts with domain I of EF-Tu (Jonak et al., 1979 Duffy et al., 1981). Thus His-66 can be crosslinked to N -bromoacetyl-lysyl-tRNA and chemical blocking of Cys-81 prevents tRNA from binding. tRNA and short fragments of its aminoacy 1-acceptor end prevent this cysteine from reacting. This interaction site for tRNA is neither far from the assumed site for the of GTP nor far from the site of proteolytic cleavage (Arg-58) near the connection to domain III. 

In addition to nonspecific lysine labeling. Lien et al. reported site-specific incorporation of a fluorescent tag into nascent proteins using a Cys-tRNA . After aminoacylation of E. coli Cys-tRNA , it was modified with a SH-reactive fluorophore (BODIPY-FL). This method may prove valuable when used in conjunction with site-directed mutagenesis to create protein containing single-cysteine residues by which one can prepare the nascent protein containing single fluorophore at a defined position. 

Intrinsic proteins may be anchored in the membrane by several different mechanisms. Attachments through interactions with lipids include fatty acid acylation of the aminoacyl terminus or the formation of thiolesters with cysteine residues. In addition, a number of proteins are covalently linked to phosphatidylinositol. 

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