A catalytic amino acid and primary structure of active site in Aspergillus niger alpha-glucosidase.
The catalytic amino acid residue of Aspergillus niger alpha-glucosidase (ANGase) was identified by modification with conduritol B epoxide (CBE), a mechanism-based irreversible inactivator. The inactivation by CBE followed pseudo-first order kinetics. The interaction of CBE and ANGase conformed to a model with a reversible enzyme-inhibitor complex formed before covalent inactivation. A competitive inhibitor, Tris, decreased the inactivation rate. The incorporation of one mole of CBE per mole of ANGase was completely abolished the enzyme activity. A dissociated carboxyl group (-COO-) in the active site was suggested to attack the C-1 of CBE. ANGase was composed of two subunits (P1 and P2), of which P2 was modified by CBE. The labelled residue was included in a peptide (LY3) that was obtained from Lys-C protease digestion of CBE-bound P2. The sequence analysis of CBE-labelled LY3 showed that an Asp was the modified residue, that is, one of the catalytic amino acid residues of ANGase. The primary structure of LY3 was determined by analyzing the sequence of peptide fragments prepared by several proteases.
Kimura
A
A
Department of Applied Bioscience, Faculty of Agriculture, Hokkaido University, Sapporo, Japan.
Takata
M
M
Fukushi
Y
Y
Mori
H
H
Matsui
H
H
Chiba
S
S
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001234:Aspergillus niger / Q000201:enzymology*; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D007294:Inositol / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*
D004791:Enzyme Inhibitors; D001224:Aspartic Acid; D007294:Inositol; D000520:alpha-Glucosidases; C011423:conduritol epoxide