Protein N-terminal processing: substrate specificity of Escherichia coli and human methionine aminopeptidases

Biochemistry. 2010 Jul 6;49(26):5588-99. doi: 10.1021/bi1005464.

Abstract

Methionine aminopeptidase (MetAP) catalyzes the hydrolytic cleavage of the N-terminal methionine from newly synthesized polypeptides. The extent of removal of methionyl from a protein is dictated by its N-terminal peptide sequence. Earlier studies revealed that MetAPs require amino acids containing small side chains (e.g., Gly, Ala, Ser, Cys, Pro, Thr, and Val) as the P1' residue, but their specificity at positions P2' and beyond remains incompletely defined. In this work, the substrate specificities of Escherichia coli MetAP1, human MetAP1, and human MetAP2 were systematically profiled by screening against a combinatorial peptide library and kinetic analysis of individually synthesized peptide substrates. Our results show that although all three enzymes require small residues at the P1' position, they have differential tolerance for Val and Thr at this position. The catalytic activity of human MetAP2 toward Met-Val peptides is consistently 2 orders of magnitude higher than that of MetAP1, suggesting that MetAP2 is responsible for processing proteins containing N-terminal Met-Val and Met-Thr sequences in vivo. At positions P2'-P5', all three MetAPs have broad specificity but are poorly active toward peptides containing a proline at the P2' position. In addition, the human MetAPs disfavor acidic residues at the P2'-P5' positions. The specificity data have allowed us to formulate a simple set of rules that can reliably predict the N-terminal processing of E. coli and human proteins.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Aminopeptidases / metabolism*
  • Catalysis
  • Escherichia coli / metabolism
  • Glycoproteins / metabolism
  • Humans
  • Methionine / metabolism*
  • Methionyl Aminopeptidases
  • Peptide Library
  • Protein Processing, Post-Translational*
  • Substrate Specificity

Substances

  • Glycoproteins
  • Peptide Library
  • Methionine
  • Aminopeptidases
  • METAP1 protein, human
  • METAP2 protein, human
  • Methionyl Aminopeptidases