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Carboxyl groups and tryptophan residues in the active site of Rhizopus niveus glucoamylase
Author(s) -
Inokuchi Ritsuko
Publication year - 1999
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/(sici)1521-4028(199912)39:5/6<311::aid-jobm311>3.0.co;2-b
Subject(s) - carbodiimide , active site , chemistry , maltose , tryptophan , enzyme , stereochemistry , chemical modification , biochemistry , amino acid
Functionally important carboxyl groups in the glucoamylase, Gluc1, from Rhizopus niveus were investigated by site‐specific modification using water‐soluble carbodiimide, 1‐ethyl‐3‐(3‐di‐methylaminopropyl)‐carbodiimide, and nucleophiles. A total of 7.5 carboxyl groups of the 37 present in Gluc1 were substituted in the presence of the substrate maltose, and there was a slight loss of enzy‐matic activity. After removal of maltose, re‐treatment of the deprotected enzyme reduced its activity to 3% with the modification of 1.2 carboxyl groups. It is conceivable that there is only one carboxyl group located in the active site. Fluorescence spectra of the enzyme suggested an interaction of tryptophan residues and carboxyl groups at the locus of the enzyme active site. Distinctive disruption of the protein structure in the modified enzyme was excluded by CD spectroscopy and immunological investigation.