Site-directed mutagenesis of the catalytic base glutamic acid 400 in glucoamylase from Aspergillus niger and of tyrosine 48 and glutamine 401, both hydrogen-bonded to the gamma-carboxylate group of glutamic acid 400.
Replacement of the catalytic base Glu400 by glutamine in glucoamylase from Aspergillus niger affects both substrates ground-state binding and transition-state stabilization. Compared to those of the wild-type enzyme, Km values for maltose and maltoheptaose are 12- and 3-fold higher for the Glu400-->Gln mutant, with kcat values 35- and 60-fold lower, respectively, for the same substrates. This unusually high residual activity for a glycosylase mutant at a putative catalytic group is tentatively explained by a reorganization of the hydrogen bond network, using the crystal structure of the related Aspergillus awamori var. X100 glucoamylase in complex with 1-deoxynojirimycin [Harris, E. M. S., Aleshin, A. E., Firsov, L. M., & Honzatko, R. B. (1993) Biochemistry 32, 1618-1626]. Supposedly Gln400 in the mutant hydrogen bonds to the invariant Tyr48, as does Glu400 in the wild-type enzyme. For Tyr48-->Trp A. niger glucoamylase kcat is reduced 80-100-fold, while Km is increased only 2-3-fold. Gln401 also hydrogen bonds to Glu400, but its mutation to glutamic acid has only a minor effect on activity. The Tyr48-->Trp and Glu400-->Gln glucoamylases share particular features in displaying unusually high activity below pH 4.0-which reflects lack of the wild-type catalytic base function- and unusually low binding affinity at subsite 2. Both mutants have lost 13-16 kJ mol-1 in transition-state stabilization energy.(ABSTRACT TRUNCATED AT 250 WORDS)
Frandsen
T P
TP
Department of Chemistry, Carlsberg Laboratory, Copenhagen Valby, Denmark.
Dupont
C
C
Lehmbeck
J
J
Stoffer
B
B
Sierks
M R
MR
Honzatko
R B
RB
Svensson
B
B
D001234:Aspergillus niger / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002264:Carboxylic Acids / Q000378:metabolism; D002384:Catalysis; D017931:DNA Primers; D004795:Enzyme Stability; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics*; D018698:Glutamic Acid / Q000235:genetics* / Q000378:metabolism; D005973:Glutamine / Q000235:genetics* / Q000378:metabolism; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013816:Thermodynamics; D014443:Tyrosine / Q000235:genetics* / Q000378:metabolism
D002264:Carboxylic Acids; D017931:DNA Primers; D005973:Glutamine; D018698:Glutamic Acid; D014443:Tyrosine; D005087:Glucan 1,4-alpha-Glucosidase