4. ENERGY-RICH COMPOUNDS
141
matic synthesis of acyl adenylates has been reported for tryptophanylAMP (238-238c), valyl-AMP (239), and seryl-AMP (239a).
Acyl adenylates may be identified by paper chromatography (-215)
or paper ionophoresis (238), by reaction with hydroxylamine (214216), and by the activity of these compounds in appropriate enzymatic
reactions.
(2). Biosynthesis. Studies on the mechanism of acetate activation
by an enzyme purified from yeast led Berg (213, 214) to suggest that
the over-all reaction shown in Eq. 50 actually occurs in two steps (Eqs.
51 and 52).
Acetate + ATP + CoA ^± Acetyl-CoA + AMP + PP
(50)
Acetate + ATP ^ Acetyl-AMP + PP
(51)
Acetyl-AMP + CoA — Acetyl-CoA + AMP
(52)
Although it was not possible to demonstrate the net synthesis of acetylAMP via Reaction 51, chemically synthesized acetyl-AMP reacted with
the purified enzyme in a manner consistent with Reactions 51 and 52.
Thus, acetate and ATP were produced from acetyl-AMP and PP,
whereas acetyl-CoA and AMP were the products from acetyl-AMP and
CoA (213, 214). Other acyl adenylates prepared by chemical synthesis
have been shown to react with the corresponding activating enzymes
in a comparable manner (215-217, 237, 237b). Further support for the
above formulation has been provided by studies with 0
18 -labeled substrates (240, 241).
Several explanations have been proposed to account for the failure
of acyl adenylates to accumulate when the enzyme is incubated with
the acid and ATP. It has been suggested, for example, that these substances exist only as enzyme-bound complexes (214-216, 242, 243), or
that deacylation of the intermediate may occur (215, 216, 219). Recently it has been pointed out that acyl adenylates are much more "energy-rich" (by about 6 kcal./mole) than ATP and, thus, that the
equilibrium of Reaction 51 lies far to the left (47). The isolation of
small amounts of aminoacyl adenylates could be explained, therefore, in
terms of dissociation from an enzyme, inactivity of a deacylase, or a
shift in the equilibrium by use of large amounts of reactants.
Presumably, the biosynthesis of acyl adenylates from a variety of
acids proceeds by way of reactions similar to Eq. 51. Reaction 52, also,
is typical of the various acyl adenylates, except that CoA may be replaced by other acceptors.* For example, pantoyl-AMP reacts with
* While it had been postulated originally that the "activation" of C0 2 involved
carbonyl-AMP (244), which then reacted with a branched-chain fatty acid, later
work (244a) has indicated that "active C0 2 " is more probably an "energy-rich"
C0 2 -biotin complex
(244b-e).
141
matic synthesis of acyl adenylates has been reported for tryptophanylAMP (238-238c), valyl-AMP (239), and seryl-AMP (239a).
Acyl adenylates may be identified by paper chromatography (-215)
or paper ionophoresis (238), by reaction with hydroxylamine (214216), and by the activity of these compounds in appropriate enzymatic
reactions.
(2). Biosynthesis. Studies on the mechanism of acetate activation
by an enzyme purified from yeast led Berg (213, 214) to suggest that
the over-all reaction shown in Eq. 50 actually occurs in two steps (Eqs.
51 and 52).
Acetate + ATP + CoA ^± Acetyl-CoA + AMP + PP
(50)
Acetate + ATP ^ Acetyl-AMP + PP
(51)
Acetyl-AMP + CoA — Acetyl-CoA + AMP
(52)
Although it was not possible to demonstrate the net synthesis of acetylAMP via Reaction 51, chemically synthesized acetyl-AMP reacted with
the purified enzyme in a manner consistent with Reactions 51 and 52.
Thus, acetate and ATP were produced from acetyl-AMP and PP,
whereas acetyl-CoA and AMP were the products from acetyl-AMP and
CoA (213, 214). Other acyl adenylates prepared by chemical synthesis
have been shown to react with the corresponding activating enzymes
in a comparable manner (215-217, 237, 237b). Further support for the
above formulation has been provided by studies with 0
18 -labeled substrates (240, 241).
Several explanations have been proposed to account for the failure
of acyl adenylates to accumulate when the enzyme is incubated with
the acid and ATP. It has been suggested, for example, that these substances exist only as enzyme-bound complexes (214-216, 242, 243), or
that deacylation of the intermediate may occur (215, 216, 219). Recently it has been pointed out that acyl adenylates are much more "energy-rich" (by about 6 kcal./mole) than ATP and, thus, that the
equilibrium of Reaction 51 lies far to the left (47). The isolation of
small amounts of aminoacyl adenylates could be explained, therefore, in
terms of dissociation from an enzyme, inactivity of a deacylase, or a
shift in the equilibrium by use of large amounts of reactants.
Presumably, the biosynthesis of acyl adenylates from a variety of
acids proceeds by way of reactions similar to Eq. 51. Reaction 52, also,
is typical of the various acyl adenylates, except that CoA may be replaced by other acceptors.* For example, pantoyl-AMP reacts with
* While it had been postulated originally that the "activation" of C0 2 involved
carbonyl-AMP (244), which then reacted with a branched-chain fatty acid, later
work (244a) has indicated that "active C0 2 " is more probably an "energy-rich"
C0 2 -biotin complex
(244b-e).