Physicochemical characterisation of the two active site mutants Trp(52)-->Phe and Asp(55)-->Val of glucoamylase from Aspergillus niger.
Glucoamylase 1 (GA1) from Aspergillus niger is a multidomain starch hydrolysing enzyme that consists of a catalytic domain and a starch-binding domain connected by an O-glycosylated linker. The fungus also produces a truncated form without the starch-binding domain (GA2). The active site mutant Trp(52)-->Phe of both forms and the Asp(55)-->Val mutant of the GA1 form have been prepared and physicochemically characterised and compared to recombinant wild-type enzymes. The characterisation included substrate hydrolysis, inhibitor binding, denaturant stability, and thermal stability, and the consequences for the active site of glucoamylase are discussed. The circular dichroic (CD) spectra of the mutants were very similar to the wild-type enzymes, indicating that they have similar tertiary structures. The D55V GA1 mutant showed slower kinetics of hydrolysis of maltose and maltoheptaose with delta delta G(double dagger) congruent with 22 kJ mol(-1), whereas the binding of the strong inhibitor acarbose was greatly diminished by delta delta G degrees congruent with 52 kJ mol(-1). Both W52F mutant forms have almost the same stability as the wild-type enzyme, whereas the D55V GA1 mutant showed slight destabilisation both towards denaturant and heat (DSC). The difference between the CD unfolding curves recorded by near- and far-UV indicated that D55V GA1 unfolds through a molten globule intermediate.
Christensen
Trine
T
Department of Biochemistry, August Krogh Institute, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen Ø, Denmark.
Frandsen
Torben P
TP
Kaarsholm
Niels C
NC
Svensson
Birte
B
Sigurskjold
Bent W
BW
D019943:Amino Acid Substitution / Q000235:genetics*; D001224:Aspartic Acid; D001234:Aspergillus niger / Q000201:enzymology*; D001665:Binding Sites; D002151:Calorimetry; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005936:Glucans / Q000378:metabolism; D019791:Guanidine; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D009154:Mutation; D010649:Phenylalanine; D011487:Protein Conformation; D011489:Protein Denaturation; D017384:Sequence Deletion; D013379:Substrate Specificity; D014364:Tryptophan; D014633:Valine
D005936:Glucans; D001224:Aspartic Acid; C031146:maltoheptaose; D010649:Phenylalanine; D008320:Maltose; D014364:Tryptophan; D005087:Glucan 1,4-alpha-Glucosidase; D014633:Valine; D019791:Guanidine