IMMEDIATE INTERACTION BETWEEN THE NASCENT SUBUNITS AND 2 CONSERVED AMINO-ACIDS TRP(34) AND THR(206) ARE NEEDED FOR THE CATALYTIC ACTIVITY OF ASPARTYLGLUCOSAMINIDASE

Aspartylglucosaminidase (AGA, EC 3.5.1.26) is a dimeric lysosomal hydr olase involved in the degradation of glycoproteins, The synthesized pr ecursor polypeptide of AGA is rapidly activated in the endoplasmic ret iculum by proteolysis into two subunits, Expression of the alpha- and beta-subunits of AGA in separate cDNA constructs showed that independe ntly folded subunits totally lack enzyme activity, and even when co-ex pressed in vitro they fail to produce an active heterodimer of the enz yme, Both of the subunits are required for the enzyme activity, and th e immediate interaction of the subunits in the endoplasmic reticulum i s necessary for the correct folding of the dimeric enzyme molecule. Th e specific amino acid residues essential for the active site of the AG A enzyme were further analyzed by site directed mutagenesis and in vit ro expression of mutagenized constructs, Replacement of Thr(206), the most aminoterminal residue of the beta-subunit, with Ser resulted in a complete loss of enzyme activity without influencing intracellular pr ocessing or transport of the mutant polypeptide to the lysosomes, Anal ogously, replacement of the most amino-terminal tryptophan, Trp(34) wi th Phe or Ser in the alpha-subunit, resulted in a totally inactive enz yme without influencing the intracellular processing or stability of t he polypeptide, These results suggest that the catalytic center of thi s amidase is formed by the interaction of the amino-terminal parts of two subunits and requires both Trp(34) in the alpha-subunit and Thr(20 6) in the beta-subunit.