ANALYSIS OF HUMAN ACID BETA-GLUCOSIDASE BY SITE-DIRECTED MUTAGENESIS AND HETEROLOGOUS EXPRESSION

Structure/function relationships of acid beta-glucosidase, the enzyme deficient in Gaucher disease, were evaluated by characterizing the pro teins expressed from cDNAs encoding normal and mutant enzymes. Twenty- two Gaucher disease mutations or created mutations were expressed in S podoptera frugiperda (Sf9) cells and analyzed for catalytic properties , stability, inhibitor binding, and modifier interactions. Many Gauche r disease mutations encoded highly disruptive amino acid substitutions (e.g. P289L and D409V) and produced severely compromised proteins wit h very reduced activity (k(cat) < 1% of normal) and/or stability. Six mutant enzymes had sufficient catalytic activity (k(cat) approximately 5-30% of normal) for extensive studies. The highly conservative subst itutions, i.e. F216Y or S364T and V394L, led to severe, but selective, abnormalities of enzyme stability or large decreases in catalytic act ivity, respectively. The T323I, N370S, and V394L enzymes interacted ab normally with active site-directed inhibitors and localized these resi dues to the glycon binding region. Selected mutant enzymes were poorly activated by phosphatidylserine (V394L, L444P, and R463C) or by sapos in C (L444P and T323I), indicating that the enzyme sites for interacti on with these activators were within the carboxyl one-third of the enz yme. Substitutions of Ser, Glu, and/or Gly at residues Asp-443 and/or Asp-445 demonstrated important steric roles for these residues in the active site, but neither is the catalytic nucleophile. Together with p revious studies, the present analyses provide an insight into the path ogenesis of Gaucher disease and the functional organization of acid be ta-glucosidase.