Human lysosomal alpha-glucosidase. Characterization of the catalytic site.
The substrate analogue conduritol B epoxide (CBE) is demonstrated to be an active site-directed inhibitor of human lysosomal alpha-glucosidase. A competitive mode of inhibition is obtained with glycogen as natural and 4-methylumbelliferyl-alpha-D-glucopyranoside as artificial substrate. The inactivation of the enzyme is time and concentration dependent and results in the covalent binding of CBE. Catalytic activity is required for binding to occur. CBE-labeled peptides containing the catalytic residue of lysosomal alpha-glucosidase were isolated and identified by microsequencing and amino acid analysis. The peptides appeared to originate from a protein domain which is highly conserved among alpha-amylases, maltase, glucoamylases, and transglucanosylases. Based on the sequence similarity and the mechanism of CBE binding, Asp-518 is predicted to be the essential carboxylate in the active site of lysosomal alpha-glucosidase. The functional importance of Asp-518 and other residues around the catalytic site was studied by expression of in vitro mutagenized alpha-glucosidase cDNA in transiently transfected COS cells. Substitution of Asp-513 by Glu-513 is shown to interfere with the posttranslational modification and the intracellular transport of the alpha-glucosidase precursor. The residues Trp-516 and Asp-518 are demonstrated to be critical for catalytic function.
Hermans
M M
MM
Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands.
Kroos
M A
MA
van Beeumen
J
J
Oostra
B A
BA
Reuser
A J
AJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004252:DNA Mutational Analysis; D065089:Glycoside Hydrolase Inhibitors; D006801:Humans; D007294:Inositol / Q000031:analogs & derivatives* / Q000494:pharmacology; D008247:Lysosomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D013329:Structure-Activity Relationship; D000520:alpha-Glucosidases / Q000737:chemistry / Q000235:genetics
D065089:Glycoside Hydrolase Inhibitors; D009841:Oligonucleotides; D010446:Peptide Fragments; D007294:Inositol; D000520:alpha-Glucosidases; C011423:conduritol epoxide