An active-site peptide containing the second essential carboxyl group of dextransucrase 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 glycine 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 inactivated the enzyme, about 35% of the activity remained during a 36-min incubation. When 10 mol of imidazole residues/mol of the enzyme was modified 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 inactivation of the sucrose-cleaving activity. In the case of DEP, the addition of sucrose or dextran gave no influence on the inactivation of the enzyme. Therefore, the carboxyl group seemed to play a more important role in the substrate binding and in the catalytic activity of the dextransucrase than the imidazolium group. Differential labeling of Leuconostoc dextransucrase by EDC was conducted in the presence of a sucrose analog, sucrose monocaprate. The fluorescent probe N-(1-naphthyl)ethylenediamine (EDAN) was used as the nucleophile instead of GEE. A fluorescent 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Funane
K
K
Molecular Engineering Laboratory, National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Ibaraki, Japan.
Shiraiwa
M
M
Hashimoto
K
K
Ichishima
E
E
Kobayashi
M
M
D000595:Amino Acid Sequence; D001241:Aspirin / Q000031:analogs & derivatives; D001665:Binding Sites; D004789:Enzyme Activation / Q000187:drug effects; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D005456:Fluorescent Dyes; D005964:Glucosyltransferases / Q000737:chemistry*; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007934:Leuconostoc / Q000201:enzymology*; D008969:Molecular Sequence Data; D017385:Sequence Homology; D013295:Streptococcus mutans / Q000201:enzymology
D005456:Fluorescent Dyes; C022010:glycine ethyl ester; D006639:Histidine; C000954:Edan; D005964:Glucosyltransferases; C020025:dextransucrase; D001241:Aspirin; D005022:Ethyldimethylaminopropyl Carbodiimide; D005998:Glycine