Abstract
1. A large fluorescence enhancement of 2 p-toluidinylnaphthalene-6-sulfonate (TNS) observed in the presence of amylose was utilized to monitor quantitatively the time course of porcine pancreatic α-amylase [EC 3. 2. 1. 1] (PPA)-catalyzed hydrolysis of amylose with a numberaverage degree of polymerization of 16.8.
2. The slope of the plot of decrease in the relative fluorescence intensity of the TNS-amylose system (termed as the fluorescence value) versus the number of linkages hydrolyzed (reducing value) (Kondo, H. et al. (1977) Agric. Biol. Chem. 41, 631-634) in the course of PPA-catalyzed hydrolysis was shown to be useful to describe the degree of “multiple attack”, which is defined by the number of reattacks on a long chain substrate molecule per one encounter of the enzyme and the substrate. A parameter γ was defined as the ratio of the reciprocal of the slopes obtained at each pH to that at pH 10.5, where the multiple attack is not operating.
3. The γ versus pH profile gave an apparent pK value of about 9, indicating that some ionizable groups participate in the multiple attack mechanism.
4. Based on a reaction scheme involving a “sliding” of the substrate molecule on the enzyme, which may contribute to the multiple attack mechanism, besides binding, dissociation, and cleavage steps of the substrate, and on the assumption of the steady state for the enzymesubstrate complex, rate equations were obtained to describe the time course of hydrolysis of a linear substrate. The product distribution with the progress of the reaction can be calculated theoretically, and is dependent on the number of multiple attack and the mode of sliding. The number of multiple attack can be estimated from this distribution, and the fluorescence value can be calculated theoretically by combining the product distribution with the relative efficiency of fluorescence intensity of each maltooligosaccharide (Nakatani, H. et al. (1977) Biopolymers 16, 2363-2370). By comparing the experimental data with the theoretical ones, it was suggested that the multiple attack occurs through the sliding by maltose unit of the retained fragment on the enzyme, which is one of the fragments produced by the initial cleavage of the substrate molecule.
5. It was found that anions (chloride, bromide, and nitrate ions) which critically affect the enzyme activity have no effect on the degree of multiple attack.
