title: Identification of histidine residues important in the catalysis and structure of aspartyl aminopeptidase.
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 k(cat). These studies reveal an important role for histidine residues both in catalysis and in the structural integrity of DAP.
authors.affiliation: Department of Pharmacology, Box 1215, Mount Sinai School of Medicine, One Gustave L Levy Place, New York, NY 10029, USA. sherwin.wilk@mssm.edu
authors.forename: Sherwin
authors.initials: S
authors.lastname: Wilk
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Wilk
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Magnusson
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D004252:DNA Mutational Analysis; D043384:Glutamyl Aminopeptidase; D006639:Histidine / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D011817:Rabbits; D017386:Sequence Homology, Amino Acid
chemical_list: D006639:Histidine; D000626:Aminopeptidases; D043384:Glutamyl Aminopeptidase