KINETICS AND MECHANISM OF HYDROLYSIS OF PHENYL ALPHA-MALTOSIDE BY SACCHARIFYING ALPHA-AMYLASE OF BACILLUS SUBTILIS .2. DEPENDENCE OF RATES OF FORMATION OF PHENOL PHENYL ALPHA-GLUCOSIDE AND MALTOTRIOSE ON SUBSTRATE CONCENTRATION

1. The hydrolysis of phenyl α-maltoside (øM) catalyzed by crystalline saccharifying α-amylase [EC 3.2. 1. 1] of Bacillus subiilis was studied at various substrate concentrations at 25°C and pH5.4 in 0.02 m acetate buffer by thin layer chromatography, phenol reagent method and modified Somogyi-Nelson's method. The rates of formation of phenol (ø), vø, phenyl α-glucoside (øG), vøG, and maltotriose (MT), pmt, were determined, and their dependencies on the substrate concentration were investigated. The effect of pH on the rates of formation of ø and øG was examined at a fixed substrate concentration.2. In the parallel reactions forming ø and øG from øM, vø follows the Michaclis-Menten equation over the whole range of substrate concentration, s (0.12-120 mm). On the other hand, the plot of øG versus substrate concentration shows a sigmoidal curve at lower substrate concentrations (0.12-10mM), and the plot of s2/vøG versuss gives a straight line, although the rate obeys the Michaelis-Menten kinetics at higher substrate concentrations (11-120mM). The dependency of vøGVø, on s shows a saturation curve of the Michaelis-Menten type.3. The optimum pH for the processes forming phenol and phenyl α-glucoside is between 5.3 and 5.4, and the pH values, which give one half the velocity at the optimum pH, are 4.0 and 6.75 for formation of phenol, and 3.7 and 7.05 for formation of phenyl α-glucoside.4. The dependencies of vø and vøG on the substrate concentration can be explained neither by a simple mechanism of a single active center producing both phenol and phenyl α-glucoside nor by a mechanism of two active centers each forming a single set of products, phenol and maltose or phenyl α-glucoside and glucose. Besides an active site (active center), an activator site to which the substrate is bound is considered necessary for the formation of øG, while such an additional site is not necessary for the formation of phenol.5. The apparent rate of formation of maltotriose, VMT, was found to be proportional to the product of vøG and vø © 1969 BY THE JOURNAL OF BIOCHEMISTRY.