Thermosensitive mutants of Aspergillus awamori glucoamylase by random mutagenesis: inactivation kinetics and structural interpretation.
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.
Flory
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N
Department of Chemical Engineering, Iowa State University, Ames 50011.
Gorman
M
M
Coutinho
P M
PM
Ford
C
C
Reilly
P J
PJ
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000235:genetics; D006358:Hot Temperature; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D011994:Recombinant Proteins; D005087:Glucan 1,4-alpha-Glucosidase