A Mutant a-Amylase with Enhanced Activity Specific for Short Substrates

摘要

For some types of enzymes, it is assumed that the active site involves a definite number of subsites, each of which specifically interacts with a certain monomer unit in a polymeric substrate. The substrate and product specificities of enzymes are defined by their original subsite structure. If the subsite structure is changed by mutation, the mutant will show a new enzymatic specificity. We determined the subsite structure of Saccharomycopsis fibuligera a-amylase (Sfamy) and estimated the major substrate-binding residues of Sfamy. We altered the 210th lysine (K210), one of the assumed components of the major subsites, into arginine (R) and asparagine (N) by site-directed mutagenesis. Replacement of K210 by R strengthened the 7th and weakened the 8th subsite affinities. K210 was found to contribute to both the 8th and the 7th subsites. The catalytic activity of the K210R enzyme for the hydrolysis of maltose (G2) was three times higher than that of the native enzyme due to an increase in the affinity of the 7th subsite adjacent to the catalytic site, whereas the activity of the K210N enzyme for G2 was decreased to 1% of that of the native enzyme by a reduction in the 7th subsite affinity.