DAP intro
Peptidases are grouped into clans and families.
Clans are groups of families for which there is evidence of common ancestry. Each clan is identified with two letters, the first representing the catalytic type of the families included in the clan. Some families cannot yet be assigned to clans, and when a formal assignment is required, such a family is described as belonging to clan A-, C-, M-, S-, T- or U-, according to the catalytic type.
Families are grouped by their catalytic type, the first character representing the catalytic type: A, aspartic; C, cysteine; G, glutamic acid; M, metallo; S, serine; T, threonine; and U, unknown. The serine, threonine and cysteine peptidases utilise the amino acid as a nucleophile and form an acyl intermediate - these peptidases can also readily act as transferases. In the case of aspartic, glutamic and metallopeptidases, the nucleophile is an activated water molecule.
Metalloproteases are the most diverse of the four main types of protease, with more than 50 families identified to date. In these enzymes, a divalent cation, usually zinc, activates the water molecule. The metal ion is held in place by amino acid ligands, usually three in number. The known metal ligands are His, Glu, Asp or Lys and at least one other residue is required for catalysis, which may play an electrophillic role. (Panther Classification System http://www.pantherdb.org/panther/family.do?clsAccession=PTHR13683)
Of particular interest in this study is aspartyl aminopeptidase (DAP), a widely distributed and abundant cystosolic enzyme and its functions include the removal of 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. (S. Wilks., et. al 2002)
First detected in mouse brain cystosol as the major enzyme catalyzing the degradation of N-terminal acidic amino acid-containing peptides (J.A. Kelly., et, al. 1983), native DAP has a molecular mass of 440 kDa and is composed of 55 kDa subunits. Amino acids involved in the catalytic mechanism of M18 metallopeptidases are unknown and through experimental means it was hypothesized that histidines are involved in Zn2+ coordinations (S. Wilks., et. al 2002).
Given the conserved histidines, whose functions have been predicted experimentally by site directed mutagenesis, Wilk et al. (2002) we are able to report that these functions concur with the structural data which has recently been resolved. We are also able to propose a working model of the protein as a dodecameric tetrahedral structure with a cocatalytic zinc centre similar to glutamyl aminopeptidase, Russo & Baumann (2004). The unfolded protein substrate is guided through channels in each face of the structure by interacting with some of the conserved histidines (310, 320, 324) around the channel where it comes into contact with the active site at the centre (His82 & 401). One of the residues responsible for securing the structure is His313 which is located at the borders of adjacent subunits. Structural analysis of other highly conserved Glu and Asp residues shows that they cluster around the predicted active site His residues, indicating they conribute to catalytic function but also validating the the prediction that this is the location of the active site.
