Functional roles of Trp337 and Glu632 in Clostridium glucoamylase, as determined by chemical modification, mutagenesis, and the stopped-flow method.
Chemical modification of glucoamylase (EC 3.2.1.3) from Clostridium sp. G0005 (CGA) with N-bromosuccinimide (NBS) was carried out in the presence or absence of an inhibitor, acarbose. CGA lost its catalytic activity through NBS oxidation in the absence of acarbose. The absorbance change at 280 nm suggested that acarbose protects about 2 Trp residues from NBS oxidation. We performed peptide mapping analysis to identify the protected Trp residues, and Trp321, Trp337, Trp433, and Trp569 were identified as candidates to be protected by acarbose. These 4 Trp residues were replaced by site-directed mutagenesis with Phe. The Trp337-->Phe mutant showed very weak catalytic activity, so Trp337 is proposed as an important residue for the catalytic activity. Further, we constructed a Glu632-->Gln mutant. Glu632 is the putative catalytic base. The presteady-state kinetics of the Trp337-->Phe and Glu632-->Gln mutants and the wild-type CGA were investigated using maltotriose as a substrate. The reaction of wild-type CGA can be explained as one involving three intermediates. On the other hand, the two mutants' reactions are explained by a two-step mechanism lacking the third intermediate. Trp337 and Glu632 appear to be crucial for the formation of the third intermediate in the wild-type reaction, which precedes the transition state.
Ohnishi
H
H
Department of Agricultural Chemistry, University of Tokyo, Japan.
Matsumoto
H
H
Sakai
H
H
Ohta
T
T
D020909:Acarbose; D000595:Amino Acid Sequence; D001483:Base Sequence; D001974:Bromosuccinimide / Q000494:pharmacology*; D003013:Clostridium / Q000201:enzymology*; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005971:Glutamates; D018698:Glutamic Acid; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014312:Trisaccharides / Q000494:pharmacology; D014364:Tryptophan
D005971:Glutamates; D007004:Hypoglycemic Agents; D009838:Oligodeoxyribonucleotides; D014312:Trisaccharides; D018698:Glutamic Acid; D014364:Tryptophan; D005087:Glucan 1,4-alpha-Glucosidase; D001974:Bromosuccinimide; D020909:Acarbose