Chemical modification of Penicillium 1,2-alpha-D-mannosidase by water-soluble carbodi-imide: identification of a catalytically important aspartic acid residue.
1,2-alpha-D-Mannosidase from Penicillium citrinum was inactivated by chemical modification with 1-ethyl-3-(3-dimethylamino-propyl)carbodi-imide (EDC). Most of the activity was lost after modification in the absence of a nucleophile, glycine ethyl ester. 1-Deoxymannojirimycin (dMM), a competitive inhibitor of the enzyme, showed partial protection against the inactivation. After the modification by EDC without the presence of a nucleophile, proteolytic digests of the enzyme were analysed by reversed-phase h.p.l.c. and a unique peptide was shown to decrease when dMM was present during the modification. The peptide was absent from the digests of unmodified enzyme. The amino acid sequence of the peptide (A; Ile-Gly-Pro) was identical in part with that of the adjacent peptide (B; Ile-Gly-Pro-Asp-Ser-Trp-Gly-Trp-Asp-Pro-Lys). When cholecystokinin tetrapeptide (Trp-Met-Asp-Phe-NH2) was modified by EDC alone, the modified peptide could be separated from unmodified peptide by reversed-phase h.p.i.c., and Edman degradation was stopped before the modified aspartic acid residue. This suggested that, in the enzyme, peptide A was derived from peptide B by the modification. Consequently, Asp-4 in peptide B was assumed to be masked by dMM during the modification, and to be involved in the interaction of the enzyme with its substrate.
Yoshida
T
T
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Sendai, Japan.
Maeda
K
K
Kobayashi
M
M
Ichishima
E
E
D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000032:analysis*; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology*; D007700:Kinetics; D008361:Mannosidases / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D010407:Penicillium / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification
D009842:Oligopeptides; D010446:Peptide Fragments; D017485:1-Deoxynojirimycin; D001224:Aspartic Acid; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase; D005022:Ethyldimethylaminopropyl Carbodiimide