INHIBITION OF LACTIC ACID DEHYDROGENASE

BY FLUOROPYRUVIC ACID*

BY HARRIS BUSCH AND P. V. NAIR

(From the Department of Pharmacology, University of Illinois
College of Medicine, Chicago, Illinois)

(Received for publication, March 25, 1957)

One approach to suppression of neoplastic growth is the attempt to

inhibit selectively important enzymatic reactions of tumors. The reaction
catalyzed by lactic dehydrogenase is of particular significance to the car-
bohydrate metabolism of tumors, inasmuch as glycolysis plays the pre-

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dominant role in catabolism of glucose in tumors in viva and in vitro (l-4).
Considerable information on lactic dehydrogenase has been obtained by
Straub (5), who crystallized lactic dehydrogenase of heart muscle, and
Kubowitz and Ott (6), who purified and crystallized lactic dehydrogenase
of tumors. Quastel and Wooldridge (7) studied inhibition of dehydro-
genation of lactic acid by analogues of lactic acid, even before the enzyme
was obtained in highly purified form. It is apparent that few compounds
inhibit the forward reaction in which pyruvate and DPNHI react to yield
lactate, although the reverse reaction in which lactate and diphosphopyri-
dine nucleotide react to yield DPNH and pyruvate is inhibited by a variety
of compounds. Among the few compounds which significantly inhibit
the forward reaction are oxalic (7) and oxamic acids (8). The reverse
reaction is inhibited by these compounds and also by glycolic acid, tartronic
acid, mesotartaric acid, and the sodium bisulfite addition product of
acetaldehyde (9). Meister (10) reported that lactic dehydrogenase will
attack a number of a-0x0 acids other than pyruvic acid at considerably
slower rates, but none of these analogues was reported to inhibit the en-
zymatically catalyzed reduction of pyruvate. Inasmuch as inhibition of
glycolysis in tumors would require inhibition of the forward reaction, all
the experiments in the present studies were carried out with DPNH and
pyruvate as substrates. It was found that fluoropyruvic acid exhibited a
more marked suppression of the reduction of pyruvate to lactate than the
other available inhibitors.
* These studies were aided by grants from the United States Public Health Service,
grant No. (CY-ZSSSC), and the Jane Coffin Childs Memorial Fund for Medical Re-
search. An initial report was presented before the Forty-seventh annual meeting
of the American Society of Biological Chemists at Atlantic City, April, 1956.
i The following abbreviations are used: DPNH = reduced diphosphopyridine nu-
cleotide, LDH = lactic dehydrogenase, and F-P = fluoropyruvic acid.
377
378 INHIBITION OF LACTIC DEHYDROGENASE

Materials and Methods

Lactic Dehydrogenase--The enzyme used in these studies was twice
recrystallized rabbit muscle lactic clehyclrogenase obtained from the Sigma
Chemical Company. It was stable for at least 6 months at a temperature
of 4’, and its specific activity was approximately 100 units per mg. (11).
Assay-The activity of the enzyme was determined in a Beckman DU
spectrophotometer at 340 rnp. The additions to the cuvette were made
in the following order: 2.7 ml. of 0.1 M potassium phosphate buffer at pH
7.4, 4 to 6 y of enzyme, 0.4 mg. of DPNH, 5 to 20 pmoles of the inhibitor
as the free acid, and 5 pmoles of sodium pyruvate; the final volume was

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3.0 ml. After each addition, the cuvette was capped and shaken. After
the final addition, the cuvette was placed in the light path, and the optical
density was determined at 10 to 15 second intervals.
Substrates-Sodium pyruvate was obtained from the Nutritional Bio-
chemicals Corporation, and DPNH from the Sigma Chemical Company;
0.4 mg. of DPNH in 3.0 ml. of reaction mixture produced an optical density
of approximately 1.000 at 340 rnp.
Inhibitors-Potassium oxamate was generously supplied by Dr. George
Schwert. Glyoxylic acid and the sodium bisulfite derivative of acetal-
dehyde were generously supplied by Dr. Israel Zelitch. a-Ketobutyric acid
was obtained from the Sigma Chemical Company, acetyl phosphate from
the Schwarz Laboratories, Inc., thiomalic acid from the General Chemicals
Company, and other compounds were obtained from the usual commercial
sources.
Fluoropyruvic Acid-Synthesis of fluoropyruvic acid was carried out by
the method of Blank, Mager, and Bergmann (12). The product was
chromatographed on Dowex 1 formate columns (13) and then distilled at
60’ at a pressure of 10 to 50 ~.r. The distillate solidified in the air condenser,
and the white crystals were removed manually. The purest crystalline
compound obtained melted at 85-86”, and the melting points of the dini-
trophenylhydrazone and semicarbazone were 140-142’ and 196-198”,
respectively.
CaH303F. Calculated. C 33.96, H 2.83, F 17.92
Found. “ 33.75, “ 2.90, “ 17.67

Homogenates-In procedures in which homogenates were used, 10 per

cent homogenates of liver and kidney were prepared in isotonic KC1 in
all-glass homogenizers and were added to isotonic potassium phosphate
buffers. Fluoropyruvic acid was added in various concentrations from
the side arm after the incubation progressed to the point where linearity
of oxygen uptake was apparent.
 H. BUSCH AND P. V. NAIR 379

Results
Inhibition of Lactic Dehydrogenase-The marked inhibition of activity
of lactic dehydrogenase by fluoropyruvic acid is illustrated in Fig. 1. Addi-
tion of 2 and 5 pmoles of fluoropyruvic acid to the system resulted in a 75
and a 95 per cent suppression of activity, respectively. Increasing con-
centrations of pyruvic acid did not increase the rate of the reaction. The
rate of the enzymatic reaction was approximately the same in the presence
and absence of pyruvate when 2 pmoles of fluoropyruvic acid were present
(Fig: 1). The concentrations of pyruvate and fluoropyruvic acid utilized
ranged from 1 X 1O-4 M to 2 X 1O-3 M, since these and other experiments

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.60- INHIBITION OF L.D.H.
BY FLUOROPYRUVATE (F-P)

0
60
SEZNDS
FIG. 1. Rates of oxidation of DPNH in systems containing 4 y of lactic dehy-
drogenase, 5 pmoles of Na pyruvate, and 1 mg. of DPNH in 2.8 ml. of 0.1 M phosphate
buffer at pH 7.4. In the control system the rate was linear for 60 seconds and was
suppressed 75 per cent by 2 @moles of fluoropyruvic acid and 95 per cent by 5 pmoles
of fluoropyruvic acid. In the system containing 2pmoles of fluoropyruvic acid with-
out pyruvate, the rate was equal to that of systems containing both fluoropyruvic acid
and pyruvate.

(6) indicated that high concentrations of pyruvate alone were inhibitory

to enzyme activity (Fig. 2). The maximal activity of the enzyme system
occurred at a concentration of pyruvate of 2 X 1O-3 M (Fig. 2). At the
highest concentrations tested, the inhibitory effect of pyruvic acid did not
reduce the activity of the enzyme by more than 30 per cent of the maximal
activity.
Table I compares the inhibitory effects of a variety of compounds on
the reduction of pyruvate to lactate by lactic dehydrogenase. Very few
of the compounds tested exhibited significant inhibitory effects on the
rate of the reaction. Inhibition of 15 to 45 per cent at concentrations
of 0.004 to 0.020 M was observed for P-bromopropionic acid, @-chloropro-
pionic acid, malonic acid, sodium formaldehyde bisulfite, chloroacetic acid,
a-chloropropionic acid, a-bromopropionic acid, and acetoin. Only levu-
380 INHIBITION OF LACTIC DEHYDROGENASE

linic, oxamic, and oxalic acids inhibited the reaction to 50 per cent of the
control values, and for these 50 per cent inhibition was obtained at con-
centrations of 0.036 M, 0.0008 M, and 0.0008 M, respectively. The com-
parative inhibitory effects of oxalic and fluoropyruvic acids are presented
in Fig. 3. The inhibitory effects of fluoropyruvic acid were more marked
than those of either oxalic or oxamic acid at all concentrations studied;
the concentration producing 50 per cent inhibition of activity of the enzyme
ranged from 0.0002 to 0.0004 M. The inhibitory activity of both oxamic
and oxalic acids was found to be competitive, with respect to pyruvate,
and was reversible (14). The degree of inhibition was unaffected by the
order of addition of the reactants, as contrasted to that of fluoropyruvic

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acid (see below). No significant inhibition of the rate of the reaction was
Cl607

Y
2
z
%,-
a
d PYRUVATE CONCENTRATION
a AND L&H ACTIVITY
7

036 .oi2
MOLARITY OF PYRUVATE
FIG. 2. Effect of pyruvate concentration on DPNH oxidation in systems contain-
ing DPNH and purified lactic dehydrogenase in concentrations equivalent to those
of Fig. 1.

observed in the presence of any of the other fluoro compounds studied,

including fluoroacetic acid, hydrogen fluoride, fluoride ion, and sodium
diethylfluorooxalacetate.
Nature of Inhibition Due to Fluoropyruvic Acid-Increasing the con-
centration of pyruvic acid did not increase the rate of the reaction in the
system inhibited with fluoropyruvic acid. Increasing the concentration
of the enzyme resulted in a marked increase in activity of the system after
a concentration of 20 y per 3 ml. was reached (Fig. 4). The increase of
activity with increasing enzyme concentration does not correspond exactly
to the Ackerman-Potter curve for a non-competitive inhibitor (14). After
overnight dialysis of the enzyme-inhibitor mixture against a 0.1 M phos-
phate buffer, the activity of the enzyme was restored only to 33 per cent
of control values. Accordingly, the inhibition of lactic dehydrogenase
by fluoropyruvic acid can be interpreted as non-competitive and largely
irreversible.
 H. BUSCH AND P. V. NAIR 381

TABLE I
Inhibition of Lactic Dehydrogenase by Various Compounds

aximal inhibi-
Compound Concentration m in per cent ICSO
range studied ,f control (60
seconds)
M Al
K oxamate...................................O.0008-0.00 4 80 0.0008
“ oxalate or oxalic acid. ..................... 0.0002-0.004 a7 0.0008
a-Chloropropionic acid. ...................... 0.008-0.02 38
@-Chlkwopropionic “ ..................... ..0.004-0.02 0 22
a-Bromopropionic “ ....................... 0.012-0.02 35

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&Bromopropionic (‘ ..................... ..0.004-0.0 20 18
&Iodopropionic acid. ........................ 0.004-0.016 30
a-Oximinopropionic acid ...................... 0.016 0
p-Fluoropropionic acid. ...................... 0.02 0
Chloropyruvic acid. ......................... 0.004 0
Hydroxypyruvic acid. ........................ 0.0008-0.00~ 0
Bromopyruvic acid ........................... 0.0008-0.04 8
Malonic acid. ................................ 0.004-o .020 44
Lactic acid ................................... 0.03 16
Acrylic acid .................................. 0.06 10
Acetone......................................O.lO 0
Diethylfluorooxalacetate (Na) ................ 0.004-0.012 0
Butyric acid.................................O.O 4 0
Thiomalic acid...............................O.O 4 0
Oxalacetic acid...............................O.O 4 0
Malic acid...................................O.O 8 23
Acetoin......................................0.004-0.0 4 27
Diglycolic acid ............................... 0.02 20
Chloroacetic acid. ........................... 0.0016-0.00~ 25
Acetamide...................................0.004-0.0 4 13
Fluoroacetate,Na............................0.004~.0 2 0
Acetylmercaptoacetic acid. ................... 0.02-0.04 30
Acetyl phosphate, Li ......................... 0.04 0
Acetaldehyde................................0.16 40
Thioglycolic acid. ............................ 0.02-0.04 25
Na formaldehyde bisulfite. ................... 0.004X).016 38
KH phthalate ............ .‘. .................. 0.0044.016 0
Nicotinic acid ................................ 0.02 35
Itaconic acid ................................. 0.02 0
wKetoglutaric acid. ......................... 0.02 15
Levulinic acid................................0.02-0.0 4 60 0.036
K fluoride ................................... 0.02 0
ml. ml.
Rabbit serum immunized ..................... 0.05-0.20 81.5 0.05

Mechanism of Inhibition-One of the characteristics of the inhibition of

lactic dehydrogenase by fluoropyruvic acid is the decrease of inhibitory
382 INHIBITION OF LACTIC DEHYDROGENASE

activity with time and with neutralization of the inhibitor. Addition of

the components of the reaction to a solution of inhibitor which has been
neutralized resulted in a reaction rate which was essentially the same as
that of the control system (Fig. 5). This finding may account for the
results of Avi-Dor and Mager (15), who did not, find inhibition of lactic

.6- RELATIVE INHIBITION OF L.D.H.

BY OXALATE AND FLUOROPYRUVATE

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0 I I
ml5 .003
MOLARITY OF INHIBITOR
FIG. 3. Comparative inhibition of lactic dehydrogenase by fluoropyruvic and
oxalic acids. The IC& for fluoropyruvic acid is 4.2 X 10-’ M, while that for oxalate
is 8.3 X 1(r4 M.

EFFECT OF ENZYME CONCENTRATION

““1 f /

ov I I
15 30
pg. L.D.H.
FIG. 4. Increased rate of oxidation of DPNH with increased enzyme concentration
in fluoropyruvic acid-inhibited systems compared with control system

dehydrogenase in systems to which the sodium salt of fluoropyruvic acid

was added. Even in systems markedly inhibited by fluoropyruvic acid
(Fig. 5), some increased activity of the enzyme was noted as time pro-
gressed; at 24 hours, the activity was only 25 per cent of the control value.
Addition of fresh enzyme to any of the inhibited systems after an int,erval
of 1 minute or more resulted in reduction of pyruvate equal to control
values (Fig. 5).
 H. BUSCH AND P. V. NAIR 383

A second characteristic of the inhibition is indicated in Table II. Maxi-

mal inhibition of the enzyme by fluoropyruvic acid was obtained only
when buffer, enzyme, and DPNH were incubated together before addition
of the inhibitor (Table II). If the inhibitor was added to the enzyme in
buffer before DPNH was added to the system, markedly less inhibition

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i
MINUTES
FIG. 5. Effect of increased duration of incubation of fluoropyruvic acid on rate of
DPNH oxidation by purified lactic dehydrogenase. Rate of enzymatic reaction in-
creased from 10 to 25 per cent of control values in systems treated with 5 pmoles of
fluoropyruvic acid. Addition of fresh enzyme resulted in DPNH oxidation at a
rate equal to control values. In a system containing “neutralized” fluoropyruvic
acid in place of sodium pyruvate, the rate of DPNH oxidation was equal to control
values.

TABLE II
Efects of Order of Additions on Inhibition of Enzymatic
Activity by Fluoropyruvic Acid

Optical density per minute

(a) Control. .;. . 0.310

(b) DPNH added after LDH and F-P. 0.240
(c) LDH “ “ F-P “ DPNH. 0.230
I‘ LDH “ “ . 0.030
(d) F-P “
(e) Add fresh LDH to (d), _: : : : : : : : : : 0.300

was noted; a similar result was obtained when fluoropyruvic acid and
DPNH were mixed together before addition of LDH. By contrast,
oxamic and oxalic acids inhibited the enzyme equally well, whether added
before or after DPNH. These data suggest that a stable ternary complex
is formed between enzyme, DPNH, and fluoropyruvic acid. The site of
this binding does not seem to be a sulfhydryl group, inasmuch as cysteine
exerted no protective effect against the inhibition (Table III).
384 INHIBITION OF LACTIC DEHYDROGENASE

Enzymatic SpeciJicity of Inhibition-Fluoropyruvic acid did not signifi-

cantly depress the activity of alcohol dehydrogenase in either direction of

TABLE III
Effect of Cysteine on Inhibition of LDH by Fluoropyruvic Acid

Optical density per minute

Control................................................... 0.300
F-P added after DPNH, LDH, and cysteine, 20 pmoles. 0.020
Add 26 pmoles of cysteine after above additions. 0.020
Add fresh LDH to above system. 0.295

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625
%
I
i EFFECT OF FLUOROPYRUVIC
&, ACID ON OXIDATION OF PYRUVATE
\\, AND MALATE
s \\,
B \!
h 375 * \\
s $,
$ \.\
9,
‘A
$250 - v
k

125-

o-
0 IO 20
MICROMOLES FLUOROPYRUVIC ACID
FIG. 6. Inhibition of oxidation of malate and pyruvate in homogenate systems.
To each flask was added 0.8 ml. of 10 per cent rat liver homogenate or 0.5.ml. of rat
kidney homogenate, 20 pmoles of Na pyruvate, 5 pmoles of K malate, and 30 pmoles
of KPOa buffer at pH 7.4. The final volume was made to 3.0 ml. with isotonic KCl.
When oxidation was observed to be proceeding linearly, fluoropyruvic acid was
added to the main chamber from the side arm.

the reaction. Moreover, fluoropyruvic acid did not significantly alter the
rate of reduction of oxalacetic acid by malic dehydrogenase.
Effects on Homogenate Systems-In both liver and kidney homogenate
systems, oxidation of a mixture of pyruvate and malate or acetate and
malate was progressively inhibited with increasing concentrations of
fluoropyruvic acid (Fig. 6). However, in concentrations up to 0.003 M
there was no significant inhibition of oxidation of citrate, glutamate, and
 H. BUSCH AND I’. V. NAIK 385

a-ketoglutarate in either the liver or the kidney system. At a concentra-

tion of 0.006 M fluoropyruvic acid, oxidation of malate and pyruvate was
inhibited by 90 per cent,, whereas that of citrate, cr-ketoglutarate, or gluta-
mate was inhibited 60 to 70 per cent. These data indicate that fluoro-
pyruvic acid inhibits both oxidative and reductive metabolism of pyruvate.

DISCUSSIOS

It is not possible to identify precisely the active inhibitory compound

because of the difficulty of distinguishing between the relatively inactive
neutralized compound and the active inhibitory compound. It is possible
that fluoropyruvic acid is the inhibitory compound and undergoes con-

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densation reactions on alkalization of the medium. It is also possible that
the inhibition is due to small concentrations of fluoropyruvic anhydride,
fluoropyruvic acid, the cis or trans enol isomers of fluoropyruvic acid,
para-fluoropyruvic acid, and the d and 1 optical isomers of para-fluoro-
pyruvic acid lactone. Each of these forms might be structurally affected
by alkalization of the medium, and the possibility that one form is an
ext.remely active inhibitor cannot be excluded until the various compounds
have been obtained in pure form.2
It would appear that at least two stages were involved in the inhibition
of the enzyme; the first, a more complete but partially reversible stage,
and the second, an irreversible inhibition of the enzyme. In the first stage
the enzyme-inhibitor complex may be stabilized by hydrogen bonding
between the enzyme and the fluorine of the inhibitor, while later the
inhibitor may react wit,h some part of the reaction center and form a stable
complex; i.e., it could attack a hydroxyl or amino group and become
chemically bound to the reaction center. The failure of bromopyruvic
and chloropyruvic acids to inhibit the enzyme suggests a stereospecific
requirement for inhibitory activity.
From the standpoint of the enzymatic specificity of the inhibition due to
fluoropyruvic acid, it is of interest that oxidation of a number of com-
2 The analysisof the dinitrophenylhydrazone of the neutralized fluoropyruvic acid
was essentially the same as that of the original compound. Moreover, the total keto
acid present (16) was not markedly changed. The structural similarity which exists
between the enol forms of fluoropyruvic acid, oxalic and oxamic acids, suggests that
only one form is actively inhibitory.
0 FCH 0
NC c/0
c-c C-C
/ \ / \ /-\
HO OH HO OH NH2 OH
It seems possible that the cis and trans enol forms are in equilibrium with each other
and with the keto form and that alkalization favors a non-inhibitory form.
386 INHIBITIOS OF LACTIC DEHYDROGESASE

pounds of the citric acid cycle was not suppressed by fluoropyruvic acid.
However, oxidation of malic and pyruvic and malic and acetic acids was
markedly inhibited by fluoropyruvic acid. These data suggest that fluoro-
pyruvic acid directly inhibits pyruvic oxidase and also can form fluoroacetyl
coenzyme A, which prevents the utilization of acetate by these systems.
Not only is there no citrate accumulation in these in vitro systems as has
been found with fluoroacetate, but also there is inhibition of formation
of citrate (17). It would appear that enough fluoroacetyl coenzyme A
forms to inhibit acetate utilization, but only a relatively small amount
forms fluorocitrate, and hence sufficient aconitase and other enzymes of
the citric acid cycle remain available for oxidation of the citrate which

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is formed.

SUMMARY

1. Fluoropyruvic acid is a non-competitive, partially reversible inhibitor

of lactic dehydrogenase. In concentrations of 0.0006 M, it inhibits the
activity of the enzyme by 75 per cent. Fluoropyruvic acid is approxi-
mately twice as effective an inhibitor of lactic dehydrogenase as oxalic and
oxamic acids.
2. A variety of other compounds, including a number of fluoro com-
pounds, hydroxypyruvic, chloropyruvic, and bromopyruvic acids, exhibited
little inhibition of the enzyme.
3. Fluoropyruvic acid is unstable in alkaline solution, and, after a short
period of incubation at pH 7.4, it not only exhibits no inhibition of the
enzyme but is converted to a substrate for the enzyme.
4. Fluoropyruvic acid inhibits oxidative as well as reductive metabolism
of pyruvic acid. In homogenate systems, oxidation of malate and pyru-
vate and malate and acetate was progressively inhibited by increasing
concentrations of fluoropyruvic acid ranging from 0.0006 M to 0.006 M.
Oxidations of citrate, cY-ketoglutarate, and glutamate were not significantly
inhibited at the lower concentrations of fluoropyruvic acid, and, at the
higher concentrations, were significantly less inhibited than the oxidation
of pyruvate and malate.

BIBLIOQRAPHY

1. Busch, H., Cancer Res., 16, 365 (1955).

2. Busch, H., Goldberg, M. H., and Anderson, D. C., Cancer Res., 16, 175 (1956).
3. Warburg, O., uber den Stoffwechsel der Tumoren, Berlin (1926).
4. Cori, C. F., and Cori, G. T., J. Biol. Chem., 66, 397 (1925).
5. Straub, F. B., Biochem. J., 34, 483 (1940).
6. Kubowitz, F., and Ott, P., Biochem. Z., 314, 94 (1943).
7. Quastel, J. H., and Wooldridge, W. R., Biochem. J., 22, 689 (1928).
8. Hakala, M. T., Glaid, A. J., and Schwert, G. W., Federation PTOC., 12. 213 (1953).
9. Zelitch, I., J. Biol. Chem., 214, 251 (1957).
 H. BUSCH AND P. V. NAIR 387

10. Meister, A., J. Biol. Chem., 184, 117 (1950).

Il. Kornberg, A., in Colowick, S. P., and Kaplan, N. O., Methods in enzymology,
New York, 1, 441 (1955).
12. Blank, I., Mager, J., and Bergmann, E. D., J. Chem. Sot., 2190 (1955).
13. Busch, H., Hurlbert, R. H., and Potter, V. R., J. Biol. Chem., 198, 717 (1952).
14. Ackerman, W. W., and Potter, V. R., Proc. Sot. Exp. Biol. and Med., 72, 1 (1949).
15. Avi-Dor, Y., and Mager, J., Biochem. J., 63, 613 (1956).
16. Friedemann, T. E., and Haugen, G. E., J. Biol. Chem., 147, 415 (1943).
17. Gal, E. M., Peters, R. A., and Wakelin, R. W., Biochem. J., 64, 161 (1956).

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 INHIBITION OF LACTIC ACID
DEHYDROGENASE BY
FLUOROPYRUVIC ACID
Harris Busch and P. V. Nair
J. Biol. Chem. 1957, 229:377-387.

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