title: Insights into substrate specificity and metal activation of mammalian tetrahedral aspartyl aminopeptidase.
abstract: Aminopeptidases are key enzymes involved in the regulation of signaling peptide activity. Here, we present a detailed biochemical and structural analysis of an evolutionary highly conserved aspartyl aminopeptidase called DNPEP. We show that this peptidase can cleave multiple physiologically relevant substrates, including angiotensins, and thus may play a key role in regulating neuron function. Using a combination of x-ray crystallography, x-ray absorption spectroscopy, and single particle electron microscopy analysis, we provide the first detailed structural analysis of DNPEP. We show that this enzyme possesses a binuclear zinc-active site in which one of the zinc ions is readily exchangeable with other divalent cations such as manganese, which strongly stimulates the enzymatic activity of the protein. The plasticity of this metal-binding site suggests a mechanism for regulation of DNPEP activity. We also demonstrate that DNPEP assembles into a functionally relevant tetrahedral complex that restricts access of peptide substrates to the active site. These structural data allow rationalization of the enzyme's preference for short peptide substrates with N-terminal acidic residues. This study provides a structural basis for understanding the physiology and bioinorganic chemistry of DNPEP and other M18 family aminopeptidases.
authors.affiliation: Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965, USA.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Chen
authors.forename: Erik R
authors.initials: ER
authors.lastname: Farquhar
authors.forename: Mark R
authors.initials: MR
authors.lastname: Chance
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Palczewski
authors.forename: Philip D
authors.initials: PD
authors.lastname: Kiser
mesh_terms: D000818:Animals; D020134:Catalytic Domain / Q000502:physiology; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D043384:Glutamyl Aminopeptidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008322:Mammals; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D021382:Protein Sorting Signals / Q000502:physiology; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012160:Retina / Q000201:enzymology*; D013379:Substrate Specificity / Q000187:drug effects / Q000502:physiology
chemical_list: D002614:Chelating Agents; D008670:Metals; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D008345:Manganese; D043384:Glutamyl Aminopeptidase