U. Bakir et al., CASSETTE MUTAGENESIS OF ASPERGILLUS-AWAMORI GLUCOAMYLASE NEAR ITS GENERAL ACID RESIDUE TO PROBE ITS CATALYTIC AND PH PROPERTIES, Protein engineering, 6(8), 1993, pp. 939-946
Nine single amino acid mutations in the active site of Aspergillus awa
mori glucoamylase were made by cassette mutagenesis to alter the pH de
pendence of the enzyme and to determine possible functions of the muta
ted residues. The Glu179 --> Asp mutation expressed in yeast led to a
very large decrease in k(cat) but to no change in K(m), verifying this
residue's catalytic function. Asp176 --> Glu and Glu180 --> Asp mutat
ions affected K(m) more than k(cat), implying that Asp176 and Glu180 a
re involved in substrate binding or structural integrity. The Leu177 -
-> Asp mutation decreased k(cat) only moderately, probably by changing
the position of the general acid catalytic group, and did not affect
K(m). The Trp178 --> Asp mutation greatly decreased k(cat) while incre
asing K(m), showing the importance of Trp178 in the active site. Val18
1 --> Asp and Asn182 --> Asp mutations changed kinetic values little,
suggesting that Val181 and Asn182 are of minor catalytic and structura
l importance. Finally, insertions of Asp or Gly between residues 176 a
nd 177 resulted in almost complete loss of activity, probably caused b
y destruction of the active site structure. No large changes in pH dep
endence occurred in those mutations where kinetic values could be dete
rmined, in spite of the increase in most cases of the total negative c
harge. Increases in activation energy of maltoheptaose hydrolysis in m
ost of the mutant glucoamylases suggested cleavage of individual hydro
gen bonds in enzyme-substrate complexes.