Identification of histidine residues important in the catalysis and structure of aspartyl aminopeptidase

Sherwin Wilk, Elizabeth Wilk, Ronald P. Magnusson

Research output: Contribution to journalArticlepeer-review

29 Scopus citations

Abstract

Aspartyl aminopeptidase (DAP), a widely distributed and abundant cytosolic enzyme, removes glutamyl or aspartyl residues from N-terminal acidic amino acid-containing peptides. DAP is a member of the M18 family of the MH clan of cocatalytic metallopeptidases. The human and mouse enzymes have been cloned. We have identified 8 highly homologous eukaryotic sequences that are probable aspartyl aminopeptidases. Eight histidine residues of human DAP were sequentially mutated to phenylalanine. Mutation of His94, His170, and His440 abolished enzymatic activity. His94 and His440 are postulated to be involved in binding cocatalytic zinc atoms by homology with other members of the MH clan. Mutation of His352 dramatically reduced enzyme activity. Gel-filtration analysis of the His352 mutant revealed destabilization of the quaternary structure and dissociation of the native 440-kDa enzyme. Mutation of His33 and of histidines residing in a cluster at residues 349, 359, and 363 all decreased kcat. These studies reveal an important role for histidine residues both in catalysis and in the structural integrity of DAP.

Original languageEnglish
Pages (from-to)176-183
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume407
Issue number2
DOIs
StatePublished - 2002

Keywords

  • Angiotensin II
  • Aspartyl aminopeptidase cocatalytic metallopeptidase
  • Essential histidines
  • M18 metallopeptidase
  • Peptide metabolism

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