Esposito et al.
1115
tively, depend on the appropriateness of the theoretical
kinetic model used in its calculation. For these reasons, the
use of spontaneous rate constants for ketonization and
in solutions of pyruvic acid at pH 1.3. Furthermore, the data
in Table 2 demonstrate that at 0°C the half-life for
enolization is around 10 min and, accordingly, after even 1 h
the equilibrium between keto and enol pyruvate would have
been virtually complete. Thus all of the enzyme assays car-
ried out by Coulson and Rabin (15a) actually involved the
same fraction of enolized pyruvate and, therefore, any varia-
tions in LDH inhibition in these experiments were unrelated
to the content of enol pyruvate in solution. In fact, the ther-
modynamic data in Table 3 indicate that the pyruvic acid
solutions at pH 1.0 at 0°C utilized in that study contained
mostly hydrated pyruvate.
enolization may give less ambiguous values of Kenol
=
k0,enol/k0,keto, provided, of course, that valid determinations of
the spontaneous rate constants are made under identical
reaction conditions. For pyruvic acid, k0,enol values that are
in close agreement (when corrected for fractions of
hydration) have been reported at 25°C (3a, 3b, 4a), k0,enol
=
1.2 × 10–6 s, and when this average value is compared to
k0,keto for pyruvic acid from the work of Chiang et al. (1g),
k0,keto = 1.06 × 10–2 s, it follows that Kenol = k0,enol/k0,keto
=
1.2 × 10–6 s/1.06 × 10–2 s = 1.1 × 10–4, which is in closer
agreement with the value we report in Table 3 (6.9 × 10–5, at
25°C), allowing for differences in experimental conditions.
The kinetic and thermodynamic parameters associated
with the reversible hydration and enolization of pyruvate re-
ported in this paper may be used to access the likelihood of
the keto, hydrated, and enol forms of pyruvate to serve ei-
ther as enzyme substrates or as enzyme inhibitors. Spe-
cifically, the availability of these data allow a closer look at
the experimental data determined by Coulson and Rabin
(15a).
In that report, it was concluded that the substrate inhibi-
tion observed in the LDH reduction of pyruvate by NADH
was actually due to a substance that can be removed from
pyruvate solutions by column chromatography. It was sug-
gested that the enzyme-inhibiting substance was enol
pyruvate and not 2,2-dihydroxypropionate (hydrated pyruvate)
because, at that time, it was believed that hydrated pyruvate
could not be observed at neutral values of pH. However, ac-
cording to the thermodynamic data provided in Table 3 it is
now known that there exists over 2000 times more hydrated
pyruvate than enol pyruvate at neutral pH at room tempera-
ture.
Buffer, ionic strength, and pH effects on the magnitude of
inhibition constants have been observed for LDH (16) and
for other enzymes (24a–24f). For example, whereas at neu-
tral pH a solution of 1 × 10–3 M keto pyruvate results in
27% substrate inhibition in 0.1 M phosphate buffer, the
same concentration of keto pyruvate causes only 9% sub-
strate inhibition in 0.1 M diethylmalonate buffer(16).
Schwert and Winer (25) have also observed greater substrate
inhibition in phosphate buffers than in other buffers. There-
fore we suggest that the differences Coulson observed in the
degree of substrate inhibition arising from assays in 0.2 M
imidazole and 0.2 M phosphate buffers were due to specific
interactions between the buffer components and the en-
zymes.
Somero (14a, 14b) has demonstrated that the substrate in-
hibition observed for muscle LDH isolated from the fish
Gillichtghys mirabilis is much stronger at colder tempera-
tures than at warmer temperatures. He suggested that this
phenomenon is a property of the enzyme itself. In a more re-
cent article (26), it is reported that differences in enzyme–
substrate binding and thermal stabilities of Sphyraena LDH-
A from various barracuda species are actually due to amino
acid residues outside the active site.
It is also instructive to interpret other of Coulson and
Rabin’s results (15a) in light of the kinetic data presented in
Table 2. Kinetic runs were carried out in the earlier study
(15a) using chromatographically purified solutions of
pyruvic acid to oxidize NADH in the presence of LDH at
room temperature. Prior to the introduction of NADH and
LDH to initiate the kinetic run, the pyruvate was incubated
in 0.2 M buffer solutions for 2 min. It was noted that while
substrate inhibition was not observed in 0.2 M imidazole
buffer, rather strong inhibition was noted in 0.2 M phosphate
buffer. Because of the lack of reliable kinetic data at that
time, it was tentatively concluded that the purified pyruvate
enolized rapidly in the phosphate buffer, thereby forming the
actual substance that inhibited LDH. It was argued that the
enolization was so slow in imidazole buffer that no such in-
hibition was observed. The data in Table 2, however, show
that the half-life for the equilibration between keto and enol
pyruvate is less than a second in either 0.2 M phosphate or
0.2 M imidazole buffer. Consequently, identical (equilib-
rium) concentrations of enol pyruvate would have prevailed
under the experimental conditions in the article cited (15a).
It was also stated in that article that purified pyruvic acid so-
lutions were stable at pH 1.0 for several hours at 0°C. How-
ever, according to the results we now report at the beginning
of this section, we find that measurable quantities of a
triiodide-scavenging impurity begin to form after about 1 h
However, we have been able to show that for trout muscle
LDH, the increased sensitivity of substrate inhibition with
decreasing temperatures is due, at least in part, to the equi-
librium shift that increases the fraction of hydrated pyruvate
at lower temperatures (27a–27c). Similar results were ob-
tained using purified beef muscle and beef heart isoenzymes
of LDH (16, 27a–27c) in which substrate inhibition was ob-
served to be more severe in the case of the latter. In a typical
set of experiments with beef heart LDH (16), small quanti-
ties of preincubated solutions of pyruvate (pyruvic acid)
were added to reaction mixtures containing NADH and LDH
to initiate kinetic runs in phosphate buffer at neutral pH at
5.0°C. The preincubated solutions of pyruvate (pyruvic acid)
were as follows: (i) prepared in phosphate buffer and
preincubated at 41°C (χhydrate = 0.054); (ii) prepared in phos-
phate buffer at 5.0°C (χhydrate = 0.136); (iii) prepared in HCl,
pH 4.5, at 41°C, yielding an aqueous pyruvic acid solution
(χhydrate = 0.412); (iv) prepared in HCl, pH 4.9, at 5.0°C,
yielding an aqueous pyruvic acid solution (χhydrate = 0.801).
The data in Table 4 show that increasing the initial fraction
of hydrated pyruvate in the assay results in increased sub-
strate inhibition in the enzymatic reactions. Since the four
experiments illustrated in Table 4 represent kinetic runs at
5.0°C, the kinetic differences cannot be a property of the en-
zyme alone but must, in fact, be related to the different ini-
tial compositions of the pyruvate solutions themselves (keto
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