TY - JOUR
T1 - Catalytic mechanism of fungal glucoamylase as defined by mutagenesis of asp176, glu179 and glu180 in the enzyme from Aspergillus awamori
AU - Sierks, Michael R.
AU - Ford, Clark
AU - Reilly, Peter J.
AU - Svensson, Birte
N1 - Funding Information:
The authors are grateful to Cetus Corporation forme gift of the glucoamylase gene, yeast expression vector and S.cerevisiae strain. This project was supported by a giant from the Biotechnology Council of Iowa State University.
PY - 1990/1
Y1 - 1990/1
N2 - 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 Glu180 - Gln mutation drastically increased the KM and moderately decreased the kcat with maltose and maltoheptaose, but affected isomaltose hydrolysis less. Differences 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 Glul79.
AB - 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 Glu180 - Gln mutation drastically increased the KM and moderately decreased the kcat with maltose and maltoheptaose, but affected isomaltose hydrolysis less. Differences 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 Glul79.
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U2 - 10.1093/protein/3.3.193
DO - 10.1093/protein/3.3.193
M3 - Article
C2 - 1970434
AN - SCOPUS:0025219541
SN - 1741-0126
VL - 3
SP - 193
EP - 198
JO - Protein Engineering, Design and Selection
JF - Protein Engineering, Design and Selection
IS - 3
ER -