THE JOURNAL OF BIOLOGICAL CHEMI~TRV

Vol. 236, No. 5, May 1961

Printedin U.S. A.

Oxidation of Glyoxylic Acid to Oxalic Acid

by Glycolic Acid Oxidase*Jf
K. E. RICHARDSON~ AND N. E. TOLBERT

From the Department of Agricultural Chemistry, Michigan State University, East Lansing, Michigan

(Received for publication, December 2, 1960)

Since the identification of glycolic acid oxidase in plants (1,2), procedure (1). Subsequent steps in the purification of the en-
the end products of this system have been repeatedly investi- zyme were followed exactly as described elsewhere (5,15). Prep-
gated. All enzyme preparations from plants (3-5), animals (6), aration of the enzyme from rat livers was essentially the same as
or microorganisms (7) have been reported to catalyze the oxida- from plants, and specific details have been described by Kun
tion of glycolic acid (COOH-CH20H) to glyoxylic acid (COOH- et al. (6). In this paper the activity of the enzyme was studied
CHO). Glyoxylic acid may be subsequently converted into mainly at four stages of purity: cell-free sap, ammonium sulfate
glycine (8, 9), which is the major product of this pathway in fractionation (l), alcohol fractionation (5), and as a solution of
most plant tissues in wivo (8). In vitro, glyoxylie acid may be the final purified enzyme of equivalent specific activity as pub-

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nonenzymatically oxidized to formic acid and CO2 by the HzOz lished by Frigerio and Harbury (15). The oxidation of glycolate
formed in the prior oxidation of glycolic acid (3, 5). Enzymatic was performed at pH 8.3 and of glyoxylate at pH 7.3 or 7.7 (3).
oxidation of glyoxylic acid to oxalic acid has been reported (4, Oxygen uptake was measured in a Warburg apparatus at 30”
lo), as well as an enzymatic system for converting two glyoxylic with an atmosphere of air. All reaction vessels contained in a
acid molecules to one glyceric acid (7, 11-13). By metabolism volume of 3.7 ml, 100 pmoles of potassium phosphate buffer,
experiments in tivo, it has been demonstrated that rats convert 0.4 pmole of FMN,1 5 to 12 units of catalase, designated units
glyoxylic acid to oxalic acid (9). of glycolic acid oxidase, and specified amounts of substrates and
Since crude or purified glycolic acid oxidase obtained from inhibitors. A unit of glycolic acid oxidase activity was defined
plants had been reported to be incapable of oxidizing glyoxylic as the uptake of 1 ~1 of oxygen in 10 minutes.
acid (5, 14,15), the assumption has been made that the oxidation Free organic acids were identified by chromatography with
of glyoxylic acid to oxalic acid was catalyzed by a different en- solvents of chloroform-ethanol-formic acid (49: 49: 2) (16)) bu-
zyme than glycolic acid oxidase. However, from a partially tanol-pyridine-water (3:2: 1.5) (17)) and two dimensional chro-
purified glycolic acid oxidase from tobacco leaves, Kenton and matography in water-saturated phenol followed by n-butanol-
Mann (4) isolated an end product from glyoxylic acid oxidation propionic acid-water (18).
which had the same melting point as oxalic acid. Therefore, in No differences in enzymatic oxidation were observed among
this report we have elaborated the enzymatic properties of the our glyoxylic acid preparation (3)) a commercial preparation from
oxidation of glyoxylic acid to oxalic acid. Highly purified Nutritional Biochemicals Corporation, and a preparation gen-
glycolic acid oxidase from plants and an ammonium sulfate erously provided by I. Zelitch. Glyoxylic acid was determined
preparation from rat liver readily catalyzed this reaction, and quantitatively by bisulfite titration (19) and as its 2,4-dinitro-
no evidence was obtained for a second enzyme. The oxalic acid phenylhydrazone derivative (20). The S-benzylthiuronium
product was an inhibitor of the enzyme, particularly for the chloride derivative of oxalic acid was prepared according to the
enzymatic attack on glyoxylic acid. Tris(hydroxymethyl)- method of Donleavy (21).
aminomethane buffer also inhibited the oxidation of glyoxylic
acid by glycolic acid oxidase. These two facts appear to ex- RESULTS

plain the previous conflicting data on the oxidation of glyoxylic Glyoxylate, as well as glycolate, was oxidized by plant saps
acid. Oxalic acid may regulate the activity in vivo of the ubiq- and by an ammonium sulfate fractionated enzyme from rat liver
uitous and very active glycolic acid oxidase by limiting its own (Table I). Spinach sap, which had been reported not to oxidize
production and consequently diverting most of the glyoxylic glyoxylate (5), did not vigorously attack added glyoxylate, al-
acid to other products such as glycine. though it was capable of a rapid catalysis of glycolic acid oxida-
tion with the uptake of more than an atom of oxygen per mole
EXPERIMENTAL PROCEDURE
of glycolate consumed (Fig. 1). The uptake of an atom of oxy-
Preparation of cell-free sap and ammonium sulfate fractiona- gen per mole of glycolate was equivalent to the formation of a
tions from a variety of plants was identical with the original mole of glyoxylate and a mole of HzOz which in turn had been
oxidized to water and an atom of oxygen. After this initial
*Published with the approval of the Director of the Michigan
Agricultural Experiment Station as Jouranl Article No. 2717. rapid reaction with glycolate, the spinach sap catalyzed oxygen
t This research was aided by a grant from the National Science uptake at the same rate as that which occurred when the gly-
Foundation. oxylate was added as the substrate.
f Present address, Department of Physiological Chemistry and
Pharmacology, Ohio State University, Columbus 10, Ohio. 1 The abbreviation used is: FMN, flavin mononucleotide.

1280
May 1961 K. E. Richardson and N. E. Tolbert 1281

TABLE I at pH 7.3 (Fig. 3). The Michaelis constant for glyoxylate oxida-
Oxidation of glyoxylic acid by crude enzyme from various sources tion was found to be 5.4 x 10-3. The effect of enzyme concen-
Each system contained 20 pmoles of sodium glyoxylate. tration on reaction velocity is shown in Fig. 4.
The effect of Tris buffer on the oxidation of glyoxylate is
Enzyme source rl Ot/mg N/hr
shown in Table III. At these concentrations, Tris buffer had no
effect on glycolate oxidation, but it markedly inhibited glyoxylate
Tobacco sap .................................... 75
20 oxidation. In the usual assay for glycolic acid oxidase with
Sugar beet sap ..................................
Swiss chardsap ................................. 30 glycolate, glyoxylate oxidation was almost eliminated by the
Spinachsap ..................................... 7 combination of an adverse pH of 8.3 and the concentration of
Rat liver, ammonium sulfate fraction. 57 Tris buffer which was employed.
During the enzymatic oxidation of glyoxylate there was
essentially one atom of oxygen consumed per mole of glyoxylate
oxidized (Table IV). Glyoxylate utilization was determined by
bisulfite titration and by calorimetric analysis of the 2,4-dinitro-
280 phenylhydrazone derivative of the remaining glyoxylate. The
t enzymatic nature of the oxidation was further indicated by the

50-
EFFECT

I I
OF SPINACH
ON ENZYME
I
ACTIVITY
I I
FRACTIONS

I
I
/ /
a

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MINUTES
FIG. 1. Oxidation of glycolate and glyoxylate by spinach sap.
Each Warburg vessel contained 1.0 ml of cell-free spinach sap, 20
Nmoles of substrate and buffer, FMN, and water as described in
“Experimental Procedure”.

A 4- to 5-fold increase in the rate of glyoxylate oxidation by

spinach sap was obtained after dialysis against running tap
water for 5 hours. This result suggested the presence in spinach
sap of an inhibitor of glyoxylate oxidation which was then demon-
strated to be inhibitory upon glycolic acid oxidase from other
sources (Fig. 2). Fresh spinach sap caused a 65% inhibition of
the rate of glyoxylate oxidation when added to a glycolic acid MINUTES
oxidase preparation from sugar beet leaves. Boiled spinach sap FIG. 2. Effect of spinach sap on glyoxylate oxidation by 75
or the ether-extractable acid fraction from boiled spinach sap units of alcohol-fractionated enzyme from sugar beet leaves In
all tests, 1 ml of spinach sap or an equivalent amount of an extract
caused an 80% inhibition, whereas the acid-free fraction was
of it was added. 0, no additions; A, fresh sap; A, boiled sap;
without inhibitory effect. The inhibitor could be absorbed on 0, ether-extracted acid fraction; H, nonacid- and nonether-ex-
an Amberlite IRA-400 anionic resin, or precipitated with calcium tracted fraction.
chloride from solutions made acidic with acetic acid. These
characteristics of the inhibitor of glycolic acid oxidase attack on TABLE II
glyoxylic acid suggested that it was oxalic acid, which is known Effect of oxalate on glycolie acid oxidase
to be present in spinach in concentrations of about 14 g per 100 The oxidation of 20 pmoles of each substrate in the presence or
g of dry weight (22). Chromatography of the inhibitor with absence of added oxalate was catalyzed by 75 units of the alcohol-
known oxalic acid and oxalic-U4 acid confirmed this identifica- fractionated enzyme from sugar beet leaves.
tion.
Substrate No oxalate 30 Jmoles of
The inhibitory effect of oxalate on the oxidation of substrates oxalate
for the purified glycolic acid oxidase is shown in Table II.
plO2/20 ?nin pl oz/zo min
Whereas glycolate oxidation was inhibited only slightly by the
Glycolate.. . 150 139
concentration of oxalate which was selected, the oxidation of
L-Lactate.......................... 98 44
L-lactate and c+hydroxybutyrate was decreased by over 50%
a-Hydroxybutyrate . .. . 78 33
and the oxidation of glyoxylate by about 74%. This inhibition Glyoxylate......................... 42 11
by oxalate was competitive in nature with a K; of 3.1 x 1O-3
1282 Oxidation of Glyoxylic Acid to Oxalic Acid Vol. 236, No. 5

zyme from sugar beet or spinach similarly requires FMN for

glyoxylate oxidation. In the absence of added FMN no oxygen
uptake was observed.
The production of Hz02 during the oxidation of glyoxylate
was detected in the same manner as reported for glycolate oxida-
tion (4, 5). The addition of catalase to the system which was
oxidizing either glycolate or glyoxylate caused a decrease in the
rate of oxygen utilization, but had no effect on the total oxygen
consumed at the completion of the reaction nor on the total
substrate utilized. The addition of peroxidase plus p-coumarate
as a hydrogen donor caused an increase in the total oxygen con-
sumed. Upon addition of catalase to the purified enzyme,
oxalate production was increased from the glyoxylate substrate,
and production of CO2 and formate was decreased. Thus when
Hz02 was not destroyed by catalase as it was formed during
glyoxylate oxidation, the Hz02 would nonenzymatically oxidize
i (PM) an equivalent amount of the remaining glyoxylate to CO2 and
FIG. 3. Inhibition of glyoxylate oxidation by oxalate (i). All formate. In such circumstances the total oxygen utilization
vessels contained 75 units of the purified enzyme from sugar beet would not vary, but the amount of the end products, oxalate,
leaves and 0, 10 pmoles of sodium glyoxylate; 0, 8 pmoles of COZ, and formate, would vary according to the disposition of the
sodium glyoxylate, or n , 6 pmoles of sodium glyoxylate.
HzOz.

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If sufficient FMN and phosphate buffer instead of Tris buffer
were employed, the relative rates of oxygen consumption with
glycolate and glyoxylate substrates remained constant through-
UY 80 out all purification steps during several large preparations of the
F enzyme from sugar beet leaves. After removal of the oxalate
j: inhibitor, similar results were obtained with spinach. In addi-
f 60
tion, no variation in the relative activity was observed when the
8 highly purified enzyme was allowed to undergo slowly 90% inac-
\
N 40 tivation at 0” in 0.1 M phosphate buffer at pH 8.3. The failure
0
to vary significantly the relative activity for the two substrates
< during the purification and inactivation indicated that the same
20 enzyme was oxidizing both substrates, as had been proposed
earlier (3, 4).
If glyoxylate and glycolate were metabolized by different en-
zymes, then the addition of glyoxylate to the system metaboliz-
ML. OF ENZYME ing glycolate should have stimulated the rate of oxygen uptake.
FIG. 4. Effect of enzyme concentration on glyoxylate oxidation. If the two substrates were competing for the same site or a
The purified enzyme from sugar beet leaves and 20 pmoles of sodium sterically inhibited adjacent site on the enzyme, there should
glyoxylate were used.
have been a decrease in the rate of oxygen consumption with
TABLE III glycolate as the glyoxylate concentration was increased. With
the purified enzyme preparation this decrease in rate of glycolate
Inhibition of glyoxylate oxidation by Tris buffer
oxidation upon addition of glyoxylate was readily apparent
Each sample contained Tris buffer at the pH and concentration
(Table V). The high concentration of glyoxylate required to
indicated, plus potassium phosphate buffer to make a total of 200
pmoles of buffer. The reactions were catalyzed by 75 units of the
TABLE IV
purified enzyme from spinach leaves.
Utilization of glyoxylate by glycolic acid oxidase
Tris buffer
I 0% uptake/lo min
Each sample contained 1.0 ml of enzyme preparation and 20
pmoles of sodium glyoxylate. Incubations lasted for 3 hours.
pH 7.1 pH 8.3
The conditions are described under “Experimental Procedure.”
pVd3 Pl rl
Glyoxylate consumed
0 23 18
50 17 11 Enzyme source 02 uptake
Z,CDinitrc-
100 13 4 Bisulfite
phenylhydrazine
aSSay
150 9 1 aSSay

200 7 1
Swiss Chard sap 6.2 5.8
fact that the rate of oxygen utilization was proportional to the Tobacco sap. 9.3 9.2 9.5
Spinach sap.. 1.4 2.8 2.9
concentration of enzyme present (Fig. 4).
Sugar beet (alcohol-precipi-
The FMN requirement of glycolic acid oxidase for glycolate tated)................... 12.9 14.6
oxidation has been reported previously (4, 5). The purified en-
May 1961 K. E. Richardson and N. E. Tolbert 1283

reduce the rate of oxygen uptake in the presence of glycolate TABLE V

to the rate with glyoxylate alone was indicative of the greater Effect of glyoxylate on glycolate oxidation
affinity of the enzyme for glycolate as a substrate. All samples contained sodium glycolate and sodium glyoxylate
Several tests established that oxalate was formed by the en- as indicated and either 81 units of alcohol-fractionated enzyme
zymatic oxidation of glyoxylate (Table VI). The greater por- from sugar beet leaves or 17 units of the ammonium sulfate-frac-
tion of the nonvolatile radioactive product obtained from tionated enzyme from rat liver.
complete oxidation of glycolate-2-W in the presence of excess Combined substrates Source of glycolic acid oxidase
catalase was found at the RF value of oxalic acid. Thus, oxalate-
Cl4 was a major product when dialyzed plant sap, the ammonium Glycolate Glyoxalate Sugar beet Rat liver
sulfate preparation from rat liver, or a purified glycolic acid /.lmoles pmoles pl 02/10 min plO2/60 min
oxidase from plants was used to catalyze this oxidation. The Cl4
10 0 81 103
activity coprecipitated with carrier oxalate upon addition of 10 20 65 72
calcium chloride to the solution previously adjusted to pH 5.5 10 40 57 55
with 10% acetic acid, and the Cl4 activity cochromatographed 10 60 47
with oxalic acid in several solvents. It is interesting to note 10 80 43
that in tivo or when undialyzed cell-free plant homogenates were 10 100 36
used, much of the glycolate-Cl4 was converted to glycine (8). 10 120 29
Re-examination of these experiments, however, indicated that 10 160 35
small amounts of oxalate-Cl4 were produced. 0 40 34 27
Oxalate was also isolated after almost complete oxidation of
100 mg of either glyoxylate or glycolate by incubation with the

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TABLE VI
various enzyme preparations for 10 hours. Since these experi-
Characterization of oxalate as end product
ments were run in unbuffered solutions, the acidity increased as
In a 125-ml Warburg flask, 100 pmoles of either glycolate or
oxalate was formed, so that it was necessary to readjust the pH
glyoxylate were incubated with a large amount of each enzyme
to 7.3 at 30-minute intervals with N KOH. The protein was de- preparation mentioned in “Experimental Procedure,” 4 rmoles of
natured by boiling for 5 minutes and removed by filtration. The FMN, 100 units of catalase, and water to make a final volume of
solution was adjusted to pH 5.5 with 10% acetic acid, and the 30 ml. The pH of the reaction was adjusted at 30.minute inter-
oxalate end product was precipitated with calcium chloride. vals to 7.5 for glyoxylate and 8.5 for glycolate. After near com-
The calcium oxalate was converted to the free acid by treatment pletion of oxygen uptake, the protein was removed by precipita-
with Dowex 50-H+ and identified as oxalate by chromatography. tion with heat and from each the assays were performed and
The free acid gave the characteristic blue color with diphenyl- derivatives prepared as indicated.
amine (23). Identification was verified by the melting point and Treatment Known oxalate Enzymatic end product
mixed melting point of the S-benzylthiuronium derivative (21).
CaC12, pH 5.5. . White precipi- White precipi-
DISCUSSION tate tate
The reported Michaelis constants of glycolic acid oxidase for Diphenylamine . .. Blue color Blue color
Chromatography (17)) cm
glycolate and lactate are 3.8 X 1O-4 M and 2.0 X 1O-3 M, re-
from origin. 5.8 5.4
spectively (5). On comparison with the value of 5.4 X 10e3 M
S-Benzylthiuronium de-
for the glyoxylate substrate, the affinity of the enzyme is gly- 191”
rivative, m.p.. . . . 191-193”
colate >> L-lactate > glyoxylate. 191-193” (mixed
The present data combined with previous studies (5, 15) m.p.1
indicate that the same enzyme catalyzes the oxidation of all
three of these substrates. Glyoxylate in aqueous solution exists
solely in the hydrated form, COOHCH(OH3 (24). It then is glyoxylate to glycine may dominate. In the special case of
structurally similar to an oc-hydroxy acid, and the activity of certain plants, such as spinach, oxalate accumulates to such an
the enzyme toward glyoxylate is consistent with its attack on extent that the enzyme as found in the sap from the crushed
the other or-hydroxy acids, glycolic acid, lactic acid, and oc-hy- leaves is almost completely inhibited from catalyzing glyoxylate
droxybutyric acid (1). This enzyme has always been referred oxidation. Yet when we have fed glycolate-Cl4 to the intact
to as glycolic acid oxidase, after the acid for which there is the spinach leaf, part of it was converted to oxalate. Clearly the
smallest Michaelis constant. However, its functions may be of very high oxalate concentration in spinach did not completely
significance in the conversion of lactic acid to pyruvic acid, prevent formation of still more of it.
glyoxylic acid to oxalic acid, and ol-hydroxybutyric acid to Considerable amounts of oxalate, partially as the insoluble
a-ketobutyric acid, all of which are known metabolic constituents calcium salt, exist in plant tissue, and oxalate in animals is
Metabolism studies with U4-labeled glycolate have shown that excreted in the urine. Even the slow oxidation of oxalate to CO2
it was mainly converted directly to glycine and serine in both by plants (25) is not an energy-generating system and can serve
plants (8) and animals (9). After the initial conversion of only to remove it from the tissue. Thus, oxalate in general
glycolate to glyoxylate, the glyoxylate in or near the active seems to be a nonfunctional and, in fact, undesirable end product
site of the enzyme should be particularly susceptible to further of metabolism in higher plants and animals. Oxalate formation
enzymatic oxidation to oxalate. That this does not occur to a from glyoxylate by glycolic acid oxidase may be an unnecessary
greater extent in vi00 may be in part the result of sufficient occurrence because the presence of the enzyme is needed for
inhibition of this oxidation by oxalate so that transamination of other purposes. The ubiquitous presence and the lack of ab-
1284 Oxidation of Glyoxylic Acid to Oxalic Acid Vol. 236, 50. 5

solute specificity of this enzyme suggest an evolutionary limit, so 9. WEINHOUSE, S., AND FRIEDMAN, B., J. Biol. Chem., 191, 707
far, in the development of an enzymatic site which-would be (1951).
10. JOKOBY, W. B., AND BHAT, J. V., Bacteriul. Rev., 22, 75 (1958).
specific for glycolate and which would not attack the glyoxylate 11. KRAKOW, G., AND BARKULUS, S. S., Biochim. et Biophys. Acta,
molecule of nearly similar structure. As a consequence, oxalate 21, 593 (1956).
production from glyoxylate occurs. 12. GALLELY, A. G., AND DAGLEY, S., Nature (London), 183, 1793
11959).
SUMMARY 13. QUAYLE, J. R., AND KEECH, D. B., Nature (London), 183, 1794
Glycolic acid oxidase from plants or animals catalyzed the (195,;.
14. CHIBA, H., KAWAI, F., AND UEDO, S., Bull. Research Inst.
oxidation of glyoxylate to oxalate, and no evidence for different Food Sci., Kyoto Univ., 16, 89 (1954). cf. Chem Abstr., 48,
enzymes for oxidizing glycolate and glyoxylate was obtained. 13753b (1954).
The oxidation of glyoxylate was inhibited by the oxalate end 15. FRIGERIO, N. A., AND HARBURG, H. A., J. Biol. Chem., 231,
product and tris(hydroxymethyl)aminomethane buffer. The 135 (1958).
16. STARK, J. B., GOODBAN, A. E., AND OWENS, H. S., Anal. Chem.,
affinity of the enzyme was glycolate >> L-lactate > glyoxylate.
23, 413 (1951).
In whole plants, glycolate-Cl4 was converted to oxalate-Ci4. 17. JEANES, A., WISE, G. S., AND DIMLER, R. J., Anal. Chem., 23,
415 (1951).
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 Oxidation of Glyoxylic Acid to Oxalic Acid by Glycolic Acid Oxidase
K. E. Richardson and N. E. Tolbert
J. Biol. Chem. 1961, 236:1280-1284.

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