Functional analyses of active site residues of human lysosomal aspartylglucosaminidase: implications for catalytic mechanism and autocatalytic activation.
Aspartylglucosaminidase (AGA) is a lysosomal asparaginase that participates in the breakdown of glycoproteins by cleaving the amide bond between the asparagine and the oligosaccharide chain. Active AGA is an (alphabeta)2 heterotetramer of two non-identical subunits that are cleaved proteolytically from an enzymatically inactive precursor polypeptide. On the basis of the three-dimensional structure recently determined by us, we have here mutagenized the putative active site amino acids of AGA and studied by transient expression the effect of targeted substitutions on the enzyme activity and catalytic properties of AGA. These analyses support the novel type of catalytic mechanism, suggested previously by us, in which AGA utilizes as the nucleophile the N-terminal 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 AGA precursor and putative involvement of active site amino acids in the proteolytic processing. The data obtained on the structure and function of AGA would indicate that AGA is a member of a recently described novel class of hydrolytic enzymes (amidohydrolases) sharing a common structural determinant in their three-dimensional structure and whose catalytic mechanisms with an N-terminal nucleophile seem basically to be similar.
Tikkanen
R
R
National Public Health Institute, Department of Human Molecular Genetics, Helsinki, Finland.
Riikonen
A
A
Oinonen
C
C
Rouvinen
R
R
Peltonen
L
L
D001227:Aspartylglucosylaminase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D006801:Humans; D008024:Ligands; D008247:Lysosomes / Q000201:enzymology*; D016296:Mutagenesis; D011485:Protein Binding; D011499:Protein Processing, Post-Translational
D008024:Ligands; D001227:Aspartylglucosylaminase