Mutagenesis of the Human α‐Galactosidase A Active Site, Dimer Interface, and Glycosylation Region

Abstract

Fabry disease is an X‐linked lysosomal storage disorder caused by the deficiency of the enzyme, α‐galactosidase A, which results in the accumulation of the lipid substrate. This accumulation results in obstruction of blood flow in patients and early demise at approximately 40–60 years of age. Currently approved enzyme replacement therapy using recombinant human α‐galactosidase A improves patient symptoms but storage of the toxic enzyme substrate remains in the brain and other affected organs. The structure of α‐galactosidase A has been previously determined to be a homodimer with six N‐linked glycosylation sites, and the catalytic mechanism determined to be a ping pong bi bi with the second substrate being water. The purpose of this research is to generate a more efficient enzyme replacement therapy alternative. A more efficient therapy was investigated utilizing Pichia pastoris, as well as site directed mutagenesis of the human enzyme. Three locations on the structure of α‐galactosidase A protein were targeted for mutation: the active site; the dimer interface; and the hydrophobic loop between the active site and the third glycosylation site. Viable candidates for further study into a therapeutic were determined based on catalytic efficiency (kcat/Km), thermostability, and possible glycosylation independence. Three mutations were identified, M208E (hydrophobic loop), W277C (dimer interface), and Y207W (active site) to be of potential therapeutic significance.Support or Funding InformationNIH SBIR Grants and Royalty Income From PatentsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.