Elucidation of the subsite structure of bacterial saccharifying alpha-amylase and its mode of degradation of maltose

Abstract

The subsite structure of bacterial saccharifying alpha-amylase (BSAm) was elucidated by two methods using a series of maltooligosaccharides labeled with [ 14C] d-glucose at the reducing end. The rate parameter k o K m and the cleavage frequency were obtained using the labeled substrates at sufficiently low concentrations to eliminate transglycosylation and condensation. This evaluation showed that the active center is composed of five subsites, with the catalytic site located between the 3rd and the 4th subsites from the nonreducing end. The evaluated affinity values of a subsite varied with the set of data used, which suggests some stimulation factor resulting from the chain length effect. The appearance of a time lag during the digestion of the poor substrate, maltose, was studied using radioactively labeled maltose (81.6 mM). Radioactive oligosaccharides larger than maltose were found at a significant level of more than 2% of the initial substrate in the digests, including a product peculiar to condensation, G-G ∗-G, as 8–10% of the maltotriose in the digests. This indicates that transglycosylation is a main side reaction (ca. 90%). A degradation pathway for maltose via maltosyl transfer was proposed, in which G 3 behaves as a kind of catalyst.