CASSETTE MUTAGENESIS OF ASPERGILLUS-AWAMORI GLUCOAMYLASE NEAR ITS GENERAL ACID RESIDUE TO PROBE ITS CATALYTIC AND PH PROPERTIES

Citation
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
Citations number
53
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
Journal title
ISSN journal
0269-2139
Volume
6
Issue
8
Year of publication
1993
Pages
939 - 946
Database
ISI
SICI code
0269-2139(1993)6:8<939:CMOAGN>2.0.ZU;2-#
Abstract
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.