Functional roles and subsite locations of leu177, trp178 and asn182 of Aspergillus awamori glucoamylase determined by site-directed mutagenesis

Michael Sierks, Clark Ford, Peter J. Reilly, Birte Svensson

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

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 α-(1,4)- and α-(1,6)-linked substrates indicate Leu177 is located in subsite 1. KM values obtained with the Trp178 - Arg mutation increase for an α-(1,6)-linked substrate, but not for α-(1,4)-linked substrates. Calculated differences in activation energy between substrates indicate Trp178 interacts specifically with subsite 2. The Asn182 α 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.

Original languageEnglish (US)
Pages (from-to)75-79
Number of pages5
JournalProtein Engineering, Design and Selection
Volume6
Issue number1
DOIs
StatePublished - Jan 1993
Externally publishedYes

Fingerprint

Glucan 1,4-alpha-Glucosidase
Mutagenesis
Aspergillus
Site-Directed Mutagenesis
Mutation
Substrates
Aspergillus niger
Carboxylic Acids
Amides
Catalyst activity
Activation energy
Acids
Enzymes
Carboxylic acids
Catalyst supports
Substitution reactions

Keywords

  • Catalytic mechanism
  • Glucoamylase
  • Kinetics
  • Site-directed mutagenesis
  • Transition state energy

ASJC Scopus subject areas

  • Pharmacology
  • Neuroscience(all)
  • Immunology and Microbiology(all)
  • Molecular Biology
  • Bioengineering
  • Biotechnology
  • Biochemistry

Cite this

Functional roles and subsite locations of leu177, trp178 and asn182 of Aspergillus awamori glucoamylase determined by site-directed mutagenesis. / Sierks, Michael; Ford, Clark; Reilly, Peter J.; Svensson, Birte.

In: Protein Engineering, Design and Selection, Vol. 6, No. 1, 01.1993, p. 75-79.

Research output: Contribution to journalArticle

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abstract = "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 α-(1,4)- and α-(1,6)-linked substrates indicate Leu177 is located in subsite 1. KM values obtained with the Trp178 - Arg mutation increase for an α-(1,6)-linked substrate, but not for α-(1,4)-linked substrates. Calculated differences in activation energy between substrates indicate Trp178 interacts specifically with subsite 2. The Asn182 α 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.",
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AB - 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 α-(1,4)- and α-(1,6)-linked substrates indicate Leu177 is located in subsite 1. KM values obtained with the Trp178 - Arg mutation increase for an α-(1,6)-linked substrate, but not for α-(1,4)-linked substrates. Calculated differences in activation energy between substrates indicate Trp178 interacts specifically with subsite 2. The Asn182 α 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.

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