AN ACTIVE-SITE PEPTIDE-CONTAINING THE 2ND ESSENTIAL CARBOXYL GROUP OFDEXTRANSUCRASE FROM LEUCONOSTOC-MESENTEROIDES BY CHEMICAL MODIFICATIONS

The treatment of Leuconostoc mesenteroides B-512F dextransucrase with 10 mM 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and glycin e ethyl ester (GEE) inactivated the enzyme almost completely within 24 min where the modification of one carboxyl group/mol of the enzyme by EDC was attained. Though 30 mM diethyl pyrocarbonate (DEP) also inact ivated the enzyme, about 35% of the activity remained during a 36-min incubation. When 10 mol of imidazole residues/mol of the enzyme was mo dified by DEP, 50% of the activity was still retained. The addition of the substrate sucrose greatly retarded the enzyme inactivation by EDC . However, the addition of dextran slightly protected the inactivation of the glucosyl-transferring activity and accelerated the inactivatio n of the sucrose-cleaving activity. In the case of DEP, the addition o f sucrose or dextran gave no influence on the inactivation of the enzy me. Therefore, the carboxyl group seemed to play a more important role in the substrate binding and in the catalytic activity of the dextran sucrase than the imidazolium group. Differential labeling of Leuconost oc dextransucrase by EDC was conducted in the presence of a sucrose an alog, sucrose monocaprate. The fluorescent probe N-(1-naphthyl)ethylen ediamine (EDAN) was used as the nucleophile instead of GEE. A fluoresc ent labeled peptide was isolated from a trypsin digest of the EDC-EDAN modified enzyme. The amino acid sequence of the isolated peptide was Leu-Gln-Glu-Asp-Asn-Ser-Asn-Val-Val-Val-Glu-Ala. The sequence had abou t 58% homology to those of streptococcal glucosyltransferases GTF-S an d GTF-I producing soluble and insoluble dextrans. Those peptides were distinguished from the active-site peptides of GTF-S and GTF-I from S. mutans, which contained catalytic aspartic acid residues of Asp465 an d Asp451, respectively. They were located at 30-45 amino acids toward the amino terminal from the catalytic aspartic acid. Therefore, the is olated peptide seemed to contain the second essential carboxyl group f or the catalytic activity.