Subsite Affinities of Bacterial Liquefying α-Amylase Evaluated from the Rate Parameters of Linear Substrates

1) The Michaelis constant K and molecular activity k° andor the ratio k°K for linear substrates (degree of polymerization n ranging from 3 to 320) at 25°c and pH5.85 were determined for two kinds of liquefying α-amylase [EC 3.2.1.1] from two different strains of Bacillus subtilis. Little difference in n-dependence of the rate parameters was observed between the two amylases. 2) The number of effective subsites in the active sites of the enzymes and their affinities were evaluated from the experimental results. A procedure for evaluating subsite affinities from the n-dependence of k0Km was proposed, which is applicable to the case where two or more productive complexes are involved. The values of subsite affinities of the two enzymes were found to be similar to each other. They were also similar to those obtained by Thoma et al. by the product analysis method for another liquefying α-amylase from a different strain of Bacillus subtilis. 3) Modes of cleavage of maltooligosaccharides observed qualitatively by thin layer chromatography were essentially in agreement with those predicted from the evaluated subsite affinities.