Interaction of catalytic-site mutants of Bacillus subtilis alpha-amylase with substrates and acarbose.

The interactions of the three catalytic-site mutants of Bacillus subtilis alpha-amylase/(DN176 [Asp-176----Asn], EQ208 [Glu-208----Gln] and DN269 [Asp-269----Asn]) with substrates and a pseudo-oligosaccharide inhibitor, acarbose, have been studied by means of difference absorption spectroscopy and affinity chromatography. The addition of maltopentaose or soluble starch to the inactive mutant enzymes mostly resulted in difference spectra characteristic of tryptophan perturbation, enabling determination of the dissociation constants. The results show that conversion of Glu-208 to Gln greatly enhanced substrate binding, implying that Glu-208 interacts unfavorably with the substrate's ground state, preventing its optimal fit to the active site. The affinity for acarbose was greatly reduced in DN269 and EQ208, but less so in DN176, suggesting that Asp-269 and Glu-208 are more important than Asp-176 in stabilizing the transition state. These results are consistent with Glu-208 and Asp-269 being the key catalytic residues, as proposed for Taka-amylase A.