376
PETER BERNFELD
5. Properties of and Comparison between Various a-Amylases
a. Mechanism of Enzyme Action. Because the nature of the substrate changes continuously during the action of α-amylase, the enzymesubstrate affinity varies considerably and progressively throughout the
whole α-amylatic breakdown of starch or glycogen (8), and an adequate comparison of the mechanism of action of this enzyme from different origins can be made only when the different stages of the
enzymatic reaction are considered separately. For this reason, an analysis will be made for (1) the initial stage of enzyme action involving
the original undegraded substrate; (2) the intermediate stages approximately where the molecular weight of the substrate has been reduced
considerably to produce dextrins that have just lost their property to
stain with iodine (achroic point), but which can still be further broken
down by the enzyme; and (3) the final stage of the enzymatic action at
which the rate of reaction has practically come to a standstill, leaving
behind the end product of the degradation.
(1). The initial stage of enzyme action. This phase is best characterized by the ratio of saccharifying to dextrinizing activity. The former
represents the increase in reducing power, the latter the change in the
iodine-staining properties of the substrate (185); both activities are
measured while less than 15% of the glucosidic bonds of the substrate
are hydrolyzed, with the use of "soluble starch" as the substrate, i.e., a
chemically degraded mixture of amylose and amylopectin whose tendency to retrogradate is extremely low. The data in Table IX clearly
show that the ratio of saccharifying to dextrinizing activity is the same
for a large number of pure α-amylases originating from the most diversified sources. This leads to the conclusion that during the initial stage of
starch degradation none of the α-amylases seem to favor or to neglect
the hydrolysis of any particular bond to a higher degree than the other
α-amylases from different origins; in other words, the initial mechanism
of attack appears to be the same for all α-amylases, independently of
their origin.
(2). The intermediate stages of a-amylase action. In a study on the
action of several α-amylases upon amylose, Kung et al. (28) came to the
conclusion that α-amylases from different sources differ markedly in their
action. There is no doubt that these data, shown in column 3 of Table
IX, are correct and that the phenomena observed are real. More than
one interpretation of these data is possible, however. It appears to the
writer that owing to the extremely high tendency of amylose to retrogradate, a portion of the substrate may escape enzymatic degradation for
some time because of the formation of enzyme-resistant substrate and
PETER BERNFELD
5. Properties of and Comparison between Various a-Amylases
a. Mechanism of Enzyme Action. Because the nature of the substrate changes continuously during the action of α-amylase, the enzymesubstrate affinity varies considerably and progressively throughout the
whole α-amylatic breakdown of starch or glycogen (8), and an adequate comparison of the mechanism of action of this enzyme from different origins can be made only when the different stages of the
enzymatic reaction are considered separately. For this reason, an analysis will be made for (1) the initial stage of enzyme action involving
the original undegraded substrate; (2) the intermediate stages approximately where the molecular weight of the substrate has been reduced
considerably to produce dextrins that have just lost their property to
stain with iodine (achroic point), but which can still be further broken
down by the enzyme; and (3) the final stage of the enzymatic action at
which the rate of reaction has practically come to a standstill, leaving
behind the end product of the degradation.
(1). The initial stage of enzyme action. This phase is best characterized by the ratio of saccharifying to dextrinizing activity. The former
represents the increase in reducing power, the latter the change in the
iodine-staining properties of the substrate (185); both activities are
measured while less than 15% of the glucosidic bonds of the substrate
are hydrolyzed, with the use of "soluble starch" as the substrate, i.e., a
chemically degraded mixture of amylose and amylopectin whose tendency to retrogradate is extremely low. The data in Table IX clearly
show that the ratio of saccharifying to dextrinizing activity is the same
for a large number of pure α-amylases originating from the most diversified sources. This leads to the conclusion that during the initial stage of
starch degradation none of the α-amylases seem to favor or to neglect
the hydrolysis of any particular bond to a higher degree than the other
α-amylases from different origins; in other words, the initial mechanism
of attack appears to be the same for all α-amylases, independently of
their origin.
(2). The intermediate stages of a-amylase action. In a study on the
action of several α-amylases upon amylose, Kung et al. (28) came to the
conclusion that α-amylases from different sources differ markedly in their
action. There is no doubt that these data, shown in column 3 of Table
IX, are correct and that the phenomena observed are real. More than
one interpretation of these data is possible, however. It appears to the
writer that owing to the extremely high tendency of amylose to retrogradate, a portion of the substrate may escape enzymatic degradation for
some time because of the formation of enzyme-resistant substrate and