Department of Chemical Engineering, Iowa State University, Ames 50011.
Protein Eng 7: 1005-12 (1994)
Abstract
Seven thermosensitive glucoamylase mutants generated by random
mutagenesis and expressed in Saccharomyces cerevisiae were sequenced and
their inactivation kinetics were determined. Wild-type glucoamylase
expressed in S. cerevisiae was more glycosylated and more stable than
the native Aspergillus niger enzyme. All mutants had lower free energies
of inactivation than wild-type glucoamylase. In the Ala39-->Val,
Ala302-->Val and Leu410-->Phe mutants, small hydrophobic residues were
replaced by larger ones, showing that increases in size and
hydrophobicity of residues included in hydrophobic clusters were
destabilizing. The Gly396-->Ser and Gly407-->Asp mutants had very
flexible residues replaced by more rigid ones, and this probably induced
changes in the backbone conformation that destabilized the protein. The
Pro128-->Ser mutation changed a rigid residue in an alpha-helix to a
more flexible one, and destabilized the protein by increasing the
entropy of the unfolded state. The Ala residue in the Ala442-->Thr
mutation is in the highly O-glycosylated region surrounded by
hydrophilic residues, where it may be a hydrophobic anchor linking the
O-glycosylated arm to the catalytic core. It was replaced by a residue
that potentially is O-glycosylated. In five of the seven mutations,
residues that were part of hydrophobic microdomains were changed,
confirming the importance of the latter in protein stability and
structure.
Mesh Headings
Unique Identifier: 95107940
Chemical Identifiers (Names)