FUNCTIONAL ANALYSES OF ACTIVE-SITE RESIDUES OF HUMAN LYSOSOMAL ASPARTYLGLUCOSAMINIDASE - IMPLICATIONS FOR CATALYTIC MECHANISM AND AUTOCATALYTIC ACTIVATION

Aspartylglucosaminidase (AGA) is a lysosomal asparaginase that partici pates in the breakdown of glycoproteins by cleaving the amide bond bet ween the asparagine and the oligosaccharide chain, Active AGA is an (a lpha beta)(2) heterotetramer of two non-identical subunits that are cl eaved proteolytically from an enzymatically inactive precursor polypep tide. On the basis of the three-dimensional structure recently determi ned by us, we have here mutagenized the putative active site amino aci ds of AGA and studied by transient expression the effect of targeted s ubstitutions on the enzyme activity and catalytic properties of AGA, T hese analyses support the novel type of catalytic mechanism, suggested previously by us, in which AGA utilizes as the nucleophile the N-term inal residue of the beta subunit and most importantly its alpha-amino group as a base that increases the nucleophilicity of the OH group. We also provide evidence for autocatalytic activation of the inactive AG A precursor and putative involvement of active site amino acids in the proteolytic processing. The data obtained on the structure and functi on of AGA would indicate that AGA is a member of a recently described novel class of hydrolytic enzymes (amidohydrolases) sharing a common s tructural determinant in their three-dimensional structure and whose c atalytic mechanisms with an N-terminal nucleophile seem basically to b e similar.