Cassette mutagenesis of Aspergillus awamori glucoamylase near its general acid residue to probe its catalytic and pH properties.
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.
Bakir
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U
Department of Chemical Engineering, Iowa State University, Ames 50011.
Coutinho
P M
PM
Sullivan
P A
PA
Ford
C
C
Reilly
P J
PJ
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002384:Catalysis; D005818:Genetic Engineering; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D005087:Glucan 1,4-alpha-Glucosidase