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Protein Engineering vol. 6 no. 1 pp. 75-79, 1993
© 1993 Oxford University Press

RESEARCH-ARTICLE

Functional roles and subsite locations of Leu177, Trp178 and Asn182 of Aspergillus awamori glucoamylase determined by site-directed mutagenesis

Michael R. Sierks1,4, Clark Ford2, Peter J. Reilly1 and Birte Svensson3

1Department of Chemical Engineering, Iowa State University Ames, IA 50011, USA 2Department of Genetics, Iowa State University Ames, IA 50011, USA 3Department of Chemistry, Carlsberg Laboratory DK-2500 Copenhagen Valby, Denmark 4Present address: Chemical and Biochemical Engineering Department, University of Maryland Baltimore County Baltimore, MD 21228, USA

4To whom correspondence should be addressed

Fungal glucoamylases contain four conserved regions. One region from the Aspergillus niger enzyme contains three key carboxylic acid residues, the general acid catalytic group, Glu179, along with Asp176 and Glu180. Three site-directed mutations, Leu177 – His, Trp178 – Arg and Asn182 – Ala, were constructed near these acidic groups to reveal the function of other conserved residues in this region. Leu177 and Trp178 are strictly conserved among fungal glucoamylases, while an amide, predominantly Asn, always occurs at position 182. Substitutions of Leu177 or Trp178 cause significant decreases in kcat with the substrates tested. Similar increases in activation energies obtained with Leu177 – His with both {alpha}-(1,4)- and {alpha}-(1,6)-linked substrates indicate Leu177 is located in subsite 1. KM values obtained with the Trp178 – Arg mutation increase for an {alpha}-(1,6)-linked substrate, but not for {alpha}-(1,4)-linked substrates. Calculated differences in activation energy between substrates indicate Trp178 interacts specifically with subsite 2. The Asn182 {alpha} Ala mutation did not change kcat or KM values, indicating that Asn182 is not crucial for activity. These results support a mechanism for glucoamylase catalytic activity consisting of a fast substrate binding step followed by a conformational change at subsite 1 to stabilize the transition state complex.

Keywords: catalytic mechanism/glucoamylase/kinetics/site-directed mutagenesis/transition state energy

Received June 24, 1992; revised September 26, 1992; accepted September 30, 1992.


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