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 citrimum was inactivated by c hemical modification with 1-ethyl-3-(3-dimethylamino-propyl)carbodi-im ide (EDC). Most of the activity was lost after modification in the abs ence of a nucleophile, glycine ethyl ester. 1-Deoxymannojirimycin (dMM ), a competitive inhibitor of the enzyme, showed partial protection ag ainst the inactivation. After the modification by EDC without the pres ence of a nucleophile, proteolytic digests of the enzyme were analysed by reversed-phase h.p.l.c., and a unique peptide was shown to decreas e 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 adjac ent peptide (B; Ile-Gly-Pro-Asp-Ser-Trp Gly-Trp-Asp-Pro-Lys) . When ch olecystokinin tetrapeptide (Trp-Met-Asp-Phe-NH2) was modified by EDC a lone, the modified peptide could be separated from unmodified peptide by reversed-phase h.p.l.c., and Edman degradation was stopped before t he modified aspartic acid residue. This suggested that, in the enzyme, peptide A was derived from peptide B by the modification. Consequentl y, Asp-4 in peptide B was assumed to be masked by dMM during the modif ication, and to be involved in the interaction of the enzyme with its substrate.