Glucoamylase mutants with decreased Km-values for C-6 substituted isomaltosides

Glucoamylase can catalyze the hydrolysis of methyl 6R-C-alkyl α-isomaltosides. A Trp120→Phe mutant altered in the +2 binding subsite had a Km of 0.089 mM for methyl 6R-C-methyl-α-isomaltoside compared to a Km of 0.71 mM for the wild-type enzyme. This reflects an eight-fold lower Km for this substrate; however, the kcat for the mutant was decreased 200-fold compared with the wild-type glucoamylase. With increasing size of the substituent to 6R-C-ethyl and -isopropyl, Trp120→Phe and wild-type glucoamylase have similar Km values, while kcat for the mutant increases 10- and 100-fold, respectively, approaching wild-type values. Perturbation of the structural integrity around the general acid catalyst Glu179, through elimination of the Trp120 NE1 hydrogen-bond to Glu179 OE2 in Trp120→Phe glucoamylase, seems to be counteracted by the larger C-6 substituents. The apparent complementarity between enzyme and substrate analogs emphasizes the favorable impact of hydrophobic forces in protein-carbohydrate interactions. Wild-type glucoamylase and Val181→Ala/Asn182→Ala/Gly183→Lys/Ser184→His, a quadruple mutant located beyond subsite +3, essentially maintain kcat in substrates with the three different 6R-C substituents, while Km increases from 0.45 to 47.0 mM for methyl-6R-C-isopropyl α-isomaltoside. Compared with the wild-type enzyme, the quadruple mutant has 1.5–3.7-fold improved specificity (kcat/Km) for the parent and 6R-C-alkyl isomaltosides, but 10-fold lower activity for the α-(1→4)-linked maltose caused mainly by a low kcat. This mutation at distant subsites thus influences hydrolysis of disaccharides, corroborating the presence of long-range effects on the catalytic site.