The Proteomics of N-terminal Methionine Cleavage

Methionine aminopeptidase (MAP) is a ubiquitous, essential enzyme involved in protein N-terminal methionine excision. According to the generally accepted cleavage rules for MAP, this enzyme cleaves all proteins with small side chains on the residue in the second position (P1′), but many exceptions are known. The substrate specificity of Escherichia coli MAP1 was studied in vitro with a large (>120) coherent array of peptides mimicking the natural substrates and kinetically analyzed in detail. Peptides with Val or Thr at P1′ were much less efficiently cleaved than those with Ala, Cys, Gly, Pro, or Ser in this position. Certain residues at P2′, P3′, and P4′ strongly slowed the reaction, and some proteins with Val and Thr at P1′ could not undergo Met cleavage. These in vitro data were fully consistent with data for 862 E. coli proteins with known N-terminal sequences in vivo. The specificity sites were found to be identical to those for the other type of MAPs, MAP2s, and a dedicated prediction tool for Met cleavage is now available. Taking into account the rules of MAP cleavage and leader peptide removal, the N termini of all proteins were predicted from the annotated genome and compared with data obtained in vivo. This analysis showed that proteins displaying N-Met cleavage are overrepresented in vivo. We conclude that protein secretion involving leader peptide cleavage is more frequent than generally thought. Methionine aminopeptidase (MAP) is a ubiquitous, essential enzyme involved in protein N-terminal methionine excision. According to the generally accepted cleavage rules for MAP, this enzyme cleaves all proteins with small side chains on the residue in the second position (P1′), but many exceptions are known. The substrate specificity of Escherichia coli MAP1 was studied in vitro with a large (>120) coherent array of peptides mimicking the natural substrates and kinetically analyzed in detail. Peptides with Val or Thr at P1′ were much less efficiently cleaved than those with Ala, Cys, Gly, Pro, or Ser in this position. Certain residues at P2′, P3′, and P4′ strongly slowed the reaction, and some proteins with Val and Thr at P1′ could not undergo Met cleavage. These in vitro data were fully consistent with data for 862 E. coli proteins with known N-terminal sequences in vivo. The specificity sites were found to be identical to those for the other type of MAPs, MAP2s, and a dedicated prediction tool for Met cleavage is now available. Taking into account the rules of MAP cleavage and leader peptide removal, the N termini of all proteins were predicted from the annotated genome and compared with data obtained in vivo. This analysis showed that proteins displaying N-Met cleavage are overrepresented in vivo. We conclude that protein secretion involving leader peptide cleavage is more frequent than generally thought. Protein N-terminal methionine excision (NME) 1The abbreviations used are: NME, N-terminal Met excision; LPR, leader peptide removal; MAP, methionine aminopeptidase; Nva, norvaline; Aba, α-aminobutyrate. 1The abbreviations used are: NME, N-terminal Met excision; LPR, leader peptide removal; MAP, methionine aminopeptidase; Nva, norvaline; Aba, α-aminobutyrate. is an essential cotranslational process that occurs in the cytoplasm of all organisms and in the two organelles (i.e. mitochondria and plastids) displaying protein synthesis (for reviews, see Refs. 1Bradshaw R.A. Brickey W.W. Walker K.W. N-terminal processing: the methionine aminopeptidase and Nα-acetyl transferase families.Trends Biochem. Sci. 1998; 23: 263-267Abstract Full Text Full Text PDF PubMed and Protein N-terminal methionine Sci. PubMed two of methionine aminopeptidase MAP1 and of (for a see and of the methionine PubMed at MAP1 known for the type and type methionine PubMed and in the cytoplasm and MAP1 in the and Escherichia and the a peptide the to the of MAP cleavage a for PubMed of the N termini of chains to methionine PubMed peptide of the N-terminal methionine cleavage Sci. Full Text Full Text PDF PubMed MAP1 of the N termini of chains to methionine PubMed The of Protein N-terminal methionine Sci. PubMed but this process is to be the of N-terminal is that to of the proteins of undergo this The residue of the proteins from of E. PubMed The N-terminal of proteins from and PubMed The of in the N-terminal position of and proteins from Escherichia and Biochem. PubMed and that this was for peptides with an N-terminal Met residue position to the of the in PubMed and the position was the for cleavage (for reviews, see Refs. and of Escherichia coli and PubMed and Protein and Escherichia coli and and for The of is that cleavage occurs the side is small is the for Ala, Cys, Pro, and According to this cleavage is not for side This from analysis on of the protein sequences at the of N-terminal protein Biochem. PubMed was analysis of MAP in vitro with a and of the N termini of chains to methionine PubMed N-terminal in Sci. PubMed of the methionine from of the Escherichia coli methionine aminopeptidase and PubMed of two proteins in E. coli was used to the analysis of N-terminal methionine in E. PubMed of N-terminal methionine excision from Escherichia coli proteins is the of the Sci. PubMed The of that the process was than and that cleavage was with or of protein for of protein PubMed at P1′ Methionine or not methionine at the of a PubMed This is fully the analysis of many and of the methionine PubMed for the type and type methionine PubMed was found to be for Ala, Thr Pro, and Val was less for the substrates and The is that the into the P1′ side is small and side chains with the two were on two the was the protein sequences at position These the of of the process (for a see 1Bradshaw R.A. Brickey W.W. Walker K.W. N-terminal processing: the methionine aminopeptidase and Nα-acetyl transferase families.Trends Biochem. Sci. 1998; 23: 263-267Abstract Full Text Full Text PDF PubMed see in Methionine a of the of the in Escherichia PubMed is used for analysis of the process in and in and for and for of PubMed analysis in E. coli the predicted and of proteins in the genome of Escherichia coli PubMed in Escherichia coli 1998; PubMed a and the N now for the of a of 862 the proteins in the E. coli the of this analysis the predicted and of proteins in the genome of Escherichia coli PubMed with the rules of proteins with and Ser at the that those with Gly, Pro, and Thr in this position proteins with Val at P1′ were found to is to the rules of N-terminal cleavage on a of all is proteins with Gly, Pro, Cys, or at P1′ are cleaved some of the rules for MAP cleavage to be or other protein or to analysis in vivo. This for of the proteins in the and the of proteins of the of the N of This was in the of from E. coli is PubMed of of the N termini of chains to methionine PubMed N-terminal in Sci. 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