TY - JOUR
T1 - Structure-function relationships in glucoamylases encoded by variant Saccharomycopsis fibuligera genes
AU - Solovicová, Adriana
AU - Christensen, Trine
AU - Hostinová, Eva
AU - Gašperík, Juraj
AU - Ševčík, Jozef
AU - Svensson, Birte
PY - 1999/9/15
Y1 - 1999/9/15
N2 - The mutation Gly467→Ser in Glu glucoamylase was designed to investigate differences between two highly homologous wild-type Saccharomycopsis fibuligera Gla and Glu glucoamylases. Gly467, localized in the conserved active site region, S5, is replaced by Ser in the Gla glucoamylase. These amino acid residues are the only two known to occupy this position in the elucidated glucoamylase sequences. The data from the kinetic analysis revealed that replacement of Gly467 with Ser in Glu glucoamylase decreased the k(cat) towards all substrates tested to values comparable with those of the Gla enzyme. Moreover, the mutant glucoamylase appeared to be less stable compared to the wild-type Glu glucoamylase with respect to thermal unfolding. Microcalorimetric titration studies of the interaction with the inhibitor acarbose indicated differences in the binding between Gla and Glu enzymes. The Gla glucoamylase, although less active, binds acarbose stronger (K(a) ≃ 1013·M-1) than the Glu enzyme (K(a) ≃ 1012·M-1). In all enzymes studied, the binding of acarbose was clearly driven by enthalpy, with a slightly favorable entropic contribution. The binding of another glucoamylase inhibitor, 1-deoxynojirimycin, was about 8-9 orders of magnitude weaker (K(a) ≃ 104·M-1) than that of acarbose. From comparison of kinetic parameters for the nonglycosylated and glycosylated enzymes it can be deduced that the glycosylation does not play a critical role in enzymatic activity. However, results from differential scanning calorimetry demonstrate an important role of the carbohydrate moiety in the thermal stability of glucoamylase.
AB - The mutation Gly467→Ser in Glu glucoamylase was designed to investigate differences between two highly homologous wild-type Saccharomycopsis fibuligera Gla and Glu glucoamylases. Gly467, localized in the conserved active site region, S5, is replaced by Ser in the Gla glucoamylase. These amino acid residues are the only two known to occupy this position in the elucidated glucoamylase sequences. The data from the kinetic analysis revealed that replacement of Gly467 with Ser in Glu glucoamylase decreased the k(cat) towards all substrates tested to values comparable with those of the Gla enzyme. Moreover, the mutant glucoamylase appeared to be less stable compared to the wild-type Glu glucoamylase with respect to thermal unfolding. Microcalorimetric titration studies of the interaction with the inhibitor acarbose indicated differences in the binding between Gla and Glu enzymes. The Gla glucoamylase, although less active, binds acarbose stronger (K(a) ≃ 1013·M-1) than the Glu enzyme (K(a) ≃ 1012·M-1). In all enzymes studied, the binding of acarbose was clearly driven by enthalpy, with a slightly favorable entropic contribution. The binding of another glucoamylase inhibitor, 1-deoxynojirimycin, was about 8-9 orders of magnitude weaker (K(a) ≃ 104·M-1) than that of acarbose. From comparison of kinetic parameters for the nonglycosylated and glycosylated enzymes it can be deduced that the glycosylation does not play a critical role in enzymatic activity. However, results from differential scanning calorimetry demonstrate an important role of the carbohydrate moiety in the thermal stability of glucoamylase.
KW - Differential scanning calorimetry
KW - Enzyme kinetics
KW - Glucoamylase
KW - Isothermal scanning calorimetry
KW - Site-directed mutagenesis
UR - http://www.scopus.com/inward/record.url?scp=0033568132&partnerID=8YFLogxK
U2 - 10.1046/j.1432-1327.1999.00634.x
DO - 10.1046/j.1432-1327.1999.00634.x
M3 - Article
C2 - 10491121
AN - SCOPUS:0033568132
SN - 0014-2956
VL - 264
SP - 756
EP - 764
JO - European Journal of Biochemistry
JF - European Journal of Biochemistry
IS - 3
ER -