Structure-function relationships in glucoamylases encoded by variant Saccharomycopsis fibuligera genes

The mutation Cly467-->Ser in Glu glucoamylase was designed to investigate d ifferences between two highly homologous wild-type Saccharomycapsis fibulig era Gla and Glu glucoamylases. Cly467, localized in the conserved active si te 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 re placement of Gly467 with Ser in Glu glucoamylase decreased the k(cat) towar ds all substrates tested to values comparable with those of the Gla enzyme. Moreover, the mutant glucoamylase appeared to be less stable compared to t he wild-type Glu glucoamylase with respect to thermal unfolding. Microcalor imetric titration studies of the interaction with the inhibitor acarbose in dicated differences in the binding between Gla and Glu enzymes. The Gla glu coamylase, although less active, binds acarbose stronger (K-a congruent to 10(13).M-1) than the Glu enzyme (K-a congruent to 10(12) M-1). In all enzym es studied, the binding of acarbose was clearly driven by enthalpy, with a slightly favorable entropic contribution. The binding of another glucoamyla se inhibitor, 1-deoxynojirimycin, was about 8-9 orders of magnitude weaker (K-a, congruent to 10(4) M-1) than that of acarbose. From comparison of kin etic parameters for the nonglycosylated and glycosylated enzymes it can be deduced that the glycosylation does not play a critical role in enzymatic a ctivity. However, results from differential scanning calorimetry demonstrat e an important role of the carbohydrate moiety in the thermal stability of glucoamylase.