Altering the substrate chain-length specificity of an α-glucosidase

Abstract

Dextran glucosidases show high sequence identity (50%) to Bacillus sp. SAM1606 α-glucosidase, which is more specific for short-chain substrates. Sequence comparison of these enzymes as well as molecular modeling studies predicted that the extension of loop 4 of the (β/α) 8-barrel fold may be responsible for the narrower specificity of SAM1606 α-glucosidase with respect to substrate chain length. Indeed, deletion mutants of SAM1606 α-glucosidase that lack this extension showed higher relative activities toward dextran and long-chain isomaltooligosaccharides. Kinetic and thermodynamic analyses of oligosaccharide hydrolysis catalyzed by SAM1606 α-glucosidase and its deletion mutants suggested that the loss of such extension(s) in loop 4 should energetically destabilize the Michaelis complexes with long-chain substrates to result in smaller differences between the activation free energies for the enzymatic hydrolyses of isomaltoheptaose and isomaltose than those observed for the wild-type enzyme. This is the reason that dextran glucosidase, whose loop 4 is shorter in length, shows broader substrate chain-length specificity than does SAM1606 α-glucosidase.