Department of Chemical Engineering, Iowa State University, Ames 50011.
Protein Eng 6: 939-46 (1993)
Abstract
Nine single amino acid mutations in the active site of Aspergillus
awamori glucoamylase were made by cassette mutagenesis to alter the pH
dependence of the enzyme and to determine possible functions of the
mutated residues. The Glu179-->Asp mutation expressed in yeast led to a
very large decrease in kcat but to no change in Km, verifying this
residue's catalytic function. Asp176-->Glu and Glu180-->Asp mutations
affected Km more than kcat, implying that Asp176 and Glu180 are involved
in substrate binding or structural integrity. The Leu177-->Asp mutation
decreased kcat only moderately, probably by changing the position of
the general acid catalytic group, and did not affect Km. The
Trp178-->Asp mutation greatly decreased kcat while increasing Km,
showing the importance of Trp178 in the active site. Val181-->Asp and
Asn182-->Asp mutations changed kinetic values little, suggesting that
Val181 and Asn182 are of minor catalytic and structural importance.
Finally, insertions of Asp or Gly between residues 176 and 177 resulted
in almost complete loss of activity, probably caused by destruction of
the active site structure. No large changes in pH dependence occurred in
those mutations where kinetic values could be determined, in spite of
the increase in most cases of the total negative charge. Increases in
activation energy of maltoheptaose hydrolysis in most of the mutant
glucoamylases suggested cleavage of individual hydrogen bonds in
enzyme-substrate complexes.
Mesh Headings
Unique Identifier: 94143349
Chemical Identifiers (Names)