Site-directed mutagenesis at the active site Trp120 of Aspergillus awamori glucoamylase.
Trp120 of Aspergillus awamori glucoamylase has previously been shown by chemical modification to be essential for activity and tentatively to be located near subsite 4 of the active site. To further test its role, restriction sites were inserted in the cloned A.awamori gene around the Trp120 coding region, and cassette mutagenesis was used to replace it with His, Leu, Phe and Tyr. All four mutants displayed 2% or less of the maximal activity (kcat) of wild-type glucoamylase towards maltose and maltoheptaose. Michaelis constants (KM) of mutants decreased 2- to 3-fold for maltose and were essentially unchanged for maltoheptaose compared with the wild type, except for a greater than 3-fold decrease for maltoheptaose with the Trp120----Tyr mutant. This mutant also bound isomaltose more strongly and had more selectivity for its hydrolysis than wild-type glucoamylase. A subsite map generated from malto-oligosaccharide substrates having 2-7 D-glucosyl residues indicated that subsites 1 and 2 had greater affinity for D-glucosyl residues in the Trp120----Tyr mutant than in wild-type glucoamylase. These results suggest that Trp120 from a distant subsite is crucial for the stabilization of the transition-state complex in subsites 1 and 2.
Sierks
M R
MR
Department of Chemical Engineering, Iowa State University, Ames 50011.
Ford
C
C
Reilly
P J
PJ
Svensson
B
B
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005800:Genes, Fungal; D005087:Glucan 1,4-alpha-Glucosidase / Q000096:biosynthesis / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D014364:Tryptophan / Q000235:genetics*
D004271:DNA, Fungal; D014364:Tryptophan; D005087:Glucan 1,4-alpha-Glucosidase