Catalytic mechanism of fungal glucoamylase as defined by mutagenesis of Asp176, Glu179 and Glu180 in the enzyme from Aspergillus awamori.
Asp176, Glu179 and Glu180 of Aspergillus awamori glucoamylase appeared by differential labeling to be in the active site. To test their functions, they were replaced by mutagenesis with Asn, Gln and Gln respectively, and kinetic parameters and pH dependencies of all enzyme forms were determined. Glu179----Gln glucoamylase was not active on maltose or isomaltose, while the kcat for maltoheptaose hydrolysis decreased almost 2000-fold and the KM was essentially unchanged from wild-type glucoamylase. The The Glu180----Gln mutation drastically increased the KM and moderately decreased the kcat with maltose and maltoheptaose, but affected isomaltose hydrolysis less. Difference in substrate activation energies between Glu180----Gln and wild-type glucoamylases indicate that Glu180 binds D-glucosyl residues in subsite 2. The Asp176----Asn substitution gave moderate increases and decreases in KM and kcat respectively, and therefore similar increases in activation energies for the three substrates. This and the differences in subsite binding energies between Asp176----Asn and wild-type glucoamylases suggest that Asp176 is near subsite 1, where it stabilizes the transition state and interacts with Trp120 at subsite 4. Glu179 and Asp176 are thus proposed as the general catalytic acid and base of pKa 5.9 and 2.7 respectively. The charged Glu180 contributes to the high pKa value of Glu179.
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; D001224:Aspartic Acid / Q000235:genetics*; D001230:Aspergillus / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004262:DNA Restriction Enzymes; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics / Q000378:metabolism*; D005936:Glucans / Q000378:metabolism; D005971:Glutamates / Q000235:genetics*; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007534:Isomaltose / Q000378:metabolism; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D012689:Sequence Homology, Nucleic Acid; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D005936:Glucans; D005971:Glutamates; D001224:Aspartic Acid; C031146:maltoheptaose; D018698:Glutamic Acid; D007534:Isomaltose; D008320:Maltose; D004262:DNA Restriction Enzymes; D005087:Glucan 1,4-alpha-Glucosidase