Immediate interaction between the nascent subunits and two conserved amino acids Trp34 and Thr206 are needed for the catalytic activity of aspartylglucosaminidase.

Aspartylglucosaminidase (AGA, EC 3.5.1.26) is a dimeric lysosomal hydrolase involved in the degradation of glycoproteins. The synthesized precursor polypeptide of AGA is rapidly activated in the endoplasmic reticulum by proteolysis into two subunits. Expression of the alpha- and beta-subunits of AGA in separate cDNA constructs showed that independently folded subunits totally lack enzyme activity, and even when co-expressed in vitro they fail to produce an active heterodimer of the enzyme. Both of the subunits are required for the enzyme activity, and the immediate interaction of the subunits in the endoplasmic reticulum is necessary for the correct folding of the dimeric enzyme molecule. The specific amino acid residues essential for the active site of the AGA enzyme were further analyzed by site-directed mutagenesis and in vitro expression of mutagenized constructs. Replacement of Thr206, the most amino-terminal residue of the beta-subunit, with Ser resulted in a complete loss of enzyme activity without influencing intracellular processing or transport of the mutant polypeptide to the lysosomes. Analogously, replacement of the most amino-terminal tryptophan, Trp34 with Phe or Ser in the alpha-subunit, resulted in a totally inactive enzyme without influencing the intracellular processing or stability of the polypeptide. These results suggest that the catalytic center of this amidase is formed by the interaction of the amino-terminal parts of two subunits and requires both Trp34 in the alpha-subunit and Thr206 in the beta-subunit.