N-terminal methionine

The molecular weights of all SERCA-type Ca " transport ATPases are in the range of 100-110 kDa. Their N-terminal sequences are similar Met-Glu-X(Ala, Asn, Glu, Asp)-X (Ala, Gly, He). The Met-Glu-X-X sequence serves as a signal for the acetylation of N-terminal methionine both in soluble and in membrane proteins [71,72]. 

The N-terminal methionine is posttranslationally acetylated [86], Similar N-term-inal sequences were found in several other proteins that contain N-acetylmethionine as the N-terminal amino acid, suggesting that the signal for acetylation may be the Met-Asp or Met-Glu sequence [71],... 

The N-terminal methionine residue of protein can also be employed for selective PEGylation using aldehyde-terminated PEG via a reductive amination reaction, because the N-terminal primary amine has a lower pAa of 7.8 than other amines such as lysines, whose pZa is 10.1 [7]. After reaction with aldehyde-terminated PEG at low pH, the resultant imine is reduced with sodium cyanoborohydrate to provide PEGylated protein (Fig. 4) [8, 9]. This technique was used for the production of Neulasta, which was approved for use by the FDA in 2002 [10]. 

Fig. 4 PEGylation at the N-terminal methionine residue. The difference in pKa. between the N-terminal amine and other amines in the protein enables site-specific PEGylation. After reaction with aldehyde-terminated PEG at low pH, reduction of the resultant imine produces PEGylated protein...

Kineret is the tradename given to a recently approved product based on the latter strategy. Indicated in the treatment of rheumatoid arthritis, the product consists of a recombinant form of the human IL-1 receptor antagonist. The 17.3 kDa, 153 amino acid product is produced in engineered E. coli and differs from the native human molecule in that it is non-glycosylated and contains an additional N-terminal methionine residue (a consequence of its prokaryotic expression system). 

Non-corrin cobalt has a number of interesting applications in the chemical industry, for example in the hydroformylation (OXO) reaction between CO, H2 and olefins. A number of non-corrin Co-containing enzymes have been described, including methionine aminopep-tidase, prolidase, nitrile hydratase and glucose isomerase. We describe the best characterized of these, namely the E. coli methionine aminopeptidase, a ubiquitous enzyme, which cleaves N-terminal methionine from newly translated polypeptide chains. The active site of the enzyme (Figure 15.13) contains two Co(II) ions that are coordinated by the side-chain atoms of five amino acid residues. The distance between the two Co2+ is similar to that between the two Zn2+ atoms in leucine aminopeptidase, and indeed the catalytic mechanism of methionine aminopeptidase shares many features with other metalloproteases, in particular leucine aminopeptidases. 

Htc-552 when expressed in E. coll undergoes spontaneous cytoplasmatic maturation (covalent haem attachment), in contrast to the usual periplasmatic maturation route for bacteria. NMR spectroscopy together with other techniques were employed to characterize Htc-552 expressed in the cytoplasm and periplasm of E. coli. It was shown that the cytoplasmatic product exhibits lower thermostability (r i = 85°C compared to the periplasmatic Tm = 90°C) because of structural anomalies in the region of the N-terminal helix, due to the retention of the N-terminal methionine and not to haem attachment errors. 

Termination Three codons (UAA, UAG and UGA) are stop codons which do not code for any amino acid but, instead of attaching to a tRNA molecule, they bind a protein release factor. When one of these factors is encountered by the ribosome, peptidyl transfer is aborted, the completed polypeptide chain released by hydrolysis and the ribosome subunits separate. The N-terminal methionine unit is then removed from the polypeptide chain. 

Filgrastim is a recombinant human G-CSF (produced in E. coli), approved for chemotherapy-induced neutropenia since 1991. Although the 18.8 kDa recombinant product is not glycosylated and contains an additional N-terminal methionine residue (due to expression in E. coli), it displays biological activity indistinguishable from native G-CSF. The product is presented in freeze-dried format and contains buffer elements as well as sorbitol and Tween as excipients. 

Protropin (rhGH, differs from human hormone only by containing an additional N-terminal methionine residue. Produced in E. coli)... 

In a recent paper (25), Bailey and Boulter have presented evidence for a single N-terminal methionine residue per (a suggested subunit of) 75,000 daltons. A single C-terminal sequence, —Tyr-Leu-Phe, was found using carboxypeptidases A and B and hydrazinolysis. Asparagine has also been reported as the N-terminal amino acid (59). 

That the N-terminal methionine is exposed to the solvent was also concluded from studies directed toward the reduction and alkylation of the disulfides of rhGH. The conditions that were found to be optimal for the derivatization of the four sulfhydryls of rhGH resulted in the production of a side reaction when the procedure was applied to the methionyl analog. This side product was identified as containing carboxymethyl-S-methionine at the amino terminal residue. 

Anakinra Anakinra is likewise indicated for the treatment of patients with rheumatoid arthritis. Anakinra is the recombinant form of human IL-1 receptor antagonist (IL-lra) and is identical to the naturally occurring, nonglycosylated form of the protein, with an additional N-terminal methionine residue. Binding of IL-lra to the IL-1R1 receptor does not initiate IL-1 mediated cell signaling,... 

Most polypeptides synthesized on ribosomes are later chemically modified. Thus the formyl group on the N-terminal methionine in polypeptides of bacteria is removed by a deformylase. In both bacteria and eukaryotes, the N-terminal methionine, sometimes along with a few additional amino acids, is removed by aminopeptidases. 

Ben-Bassat, A. (1991). Methods for removing N-terminal methionine from recombinant proteins. In Purification and Analysis of Recombinant Proteins (R. Seetharam and S. K. Sharma, eds.), pp. 147-159. Dekker, New York. 

The molecular mechanisms by which the extension of the N-terminus by the extra methionine residue destabilized recombinant a-lactalbumin remain unclear. Additional conformational entropy of the extra methionine residue in the unfolded state could account for the destabilization and unfolding-rate acceleration of the recombinant protein [22]. Ishikawa and coworkers reported the destabilization of recombinant bovine a-lactalbumin, similarly induced by the extra N-terminal methionine residue, and showed that the enthalpy change of thermal unfolding was the same for the authentic and recombinant proteins, indicating that the destabilization was caused by an entropic effect [42]. However, the destabilization by the extra methionine residue in the lysozyme homologous to a-lactalbumin was rather enthalpic and accompanied by a disruption of hydrogen-bond networks in the N-terminal region [43,44]. 

Virtually all proteins expressed in cells undergo processing to remove the N-terminal methionine residue encoded by the start codon , ATG. Cleavage of this initiation methionine from newly formed polypeptide chains is catalyzed by the enzyme methionine aminopeptidase (equation 9). 

In addition to Lys side chain acetylation, protein N-terminal can also be acetylated (59). In eukaryotic cells, the first residue Met in most proteins is cleaved by N-terminal methionine peptidase. The newly released N-terminal amino group is then acetylated. This modihcation can happen co-translationally before the mature peptide chain is released from the ribosome. The function of this modihcation in most cases is still not understood, although deletion of the genes involved in this modihcation has clear phenotypes (59). 

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