Occurrence of Mercaptopyruvate Sulfotransferase Activity
in Photosynthetic Organisms
Ahlert Schmidt
Botanisches Institut der Universität München, Menzinger Str. 67, D-8000 München 19,
Bundesrepublik Deutschland
Z. Naturforsch. 39c, 916-921 (1984); received April 6, 1984
Pyruvate, Mercaptopyruvate, Lemna, Spinacia, Pisum, Chlorella, Synechococcus,
Rhodopseudomonas
Mercaptopyruvate sulfotransferase activity catalyzes the formation o f pyruvate from mercapto­
pyruvate in the presence o f suitable reagents as acceptor. It was detected in Lemna minor,
Pisum sativum, Spinacia oleracea, Chlorella fusca, Synechococcus 6301, and Rhodopseudomonas
palustris. Best activity was detected using dithioerythritol as a thiol reagent; good activity was
obtained using mercaptoethanol, glutathione, mercaptopyruvate or sulfite as acceptor. The
pH-optimum for the Chlorella mercaptopyruvate sulfotransferase was found around 9; the
apparent Km for mercaptopyruvate was determined to 2 m M and for dithioerythritol for 5 m M
using crude Chlorella extracts. The role of this enzyme is discussed in relation to cysteine
catabolism by photosynthetic organisms.
Introduction
Plants and algae synthesize cysteine as the first
sulfur-containing am ino acid du rin g assim ilato ry
sulfate reduction [1]. C ysteine, how ever, is not only
the am ino acid needed for m e th io n in e and p ro tein
synthesis, but it m ight have o th e r functions for the
control o f cellular m etabolism as well. T his was
suggested recently for a control o f the cellu lar thioldisulfide level by the cysteine o x id atio n system
described for Synechococcus [2] and Chlorella [3].
Evidence from algae and plants has d em o n strate d
that elevated cysteine concentrations are toxic
[4, 5] suggesting that the in ternal cysteine pool
seems to be critical for a cell. Plants and algae
can regulate the internal cysteine co n cen tratio n
by degrading cysteine to sulfide and em it this
sulfide or oxidize it to sulfate [6 -1 6 ]. T he
precise m echanism (s) of cysteine d eg ra d atio n are
not fully understood so far. T herefo re reactions
leading to sulfide from cysteine or co m p o u n d s re­
lated to cysteine have been analyzed. W e will d e m ­
onstrate in this publication for the first tim e th a t
a m ercaptopyruvate sulfotransferase activity is p res­
ent in photosynthetic organism s. T he rela tio n o f this
enzyme to cysteine m etabolism is discussed.
Materials and Methods
a) Organisms
Spinacia oleracea L. and Pisum sativum L. w ere
grown in the greenhouse. L em na m inor L. was
0341-0382/84/0900-0916
$01.3 0 /0
m aintained in axenic culture according to B runold
and Schm idt [17], Chlorella fu sca strain 211 -8 b from
the algal collection of G öttingen was cu ltu red as
described earlier [18], and Synechococcus 6301
(Pasteur Institute Paris) was grown as axenic cu ltu re
as described earlier [19]. Rhodopseudomonas palustris
cells grown on thiosulfate were a generous gift o f
Prof. Dr. K nobloch (Erlangen).
b) Preparation o f enzym e extracts
1) Plant systems: 2 g o f leaf tissue was gro u n d in a
m ortar using 4 ml o f a buffer containing 0.1 m T risHCl buffer pH 8.0, 10 m M M gCl2, and 10 m M m e r­
captoethanol. T he crude extract was cleared by
centrifugation and the su p ern atan t was used as
enzyme source. 2) Algal systems: 2 g o f algal cells
(wet weight) were suspended in 4 ml o f the b u ffe r
m entioned above and broken in a french press at
12,000 psi ( l p s i ~ 7 K P a ) and cleared afterw ard s
by centrifugation; the su p ern atan t was used as th e
enzyme source. Extracts could be stored frozen
w ithout loss o f activity for several days.
c) Protein determination
The Coom assie blue m ethod o f B radford [20] was
used with the dye reagent p rep aratio n from B iorad.
Bovine serum album in was used as a reference.
d) Measurements o f mercaptopyruvate
sulfotransferase activity
This enzyme activity was d eterm in ed follow ing
the procedure according to Jarabak and W estley [21].
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A. Schmidt • Occurrence of Mercaptopyruvate Sulfotransferase Activity in Photosynthetic Organisms
917
e) Chemicals
M ercaptopyruvate was o b tain ed from F lu k a A G
(N eu-U lm , W est-G erm any); 2,4-dinitrophenylhydrazin and all o th er chem icals not m entioned w ere
obtained from M erck (D arm stad t, W est-G erm any).
^
^
CL
Results
Chlorella extracts catalyzed the form atio n o f
pyruvate from m ercaptopyruvate in the presence
of various thiols. In order to define the conditions
for the m easurem ent o f this m ercap to p y ru v ate
sulfotransferase activity, the follow ing experim ents
were perform ed, using crude Chlorella extracts
prepared as described in M aterials and M ethods.
a) pH -optim um
O
c
7
8
9
10
Fig. 1. pH-dependence of the mercaptopyruvate sulfotrans­
ferase activity from Chlorella. Conditions: Each vessel
contained (in umol) in a total volume of 1 ml: Tris-HCl:
100 (pH varied as indicated); dithioerythritol: 10; mer­
captopyruvate: 5; and 1.1 mg o f crude Chlorella extract
protein. Incubation for 30 min at 37 °C.
M aximal pyruvate form ation was achieved at pH values above 9, as can be seen from the d ata o f
Fig. 1. We have used a pH o f 9.0 for all experim ents
to be described.
b) Protein dependence o f the m ercaptopyruvate
sulfotransferase activity
The data o f Fig. 2 d em onstrate th a t good activity
o f this enzyme could be m easured in the range o f
1 mg of crude protein added. T he reaction is lin ear
at lower protein concentrations, how ever at h ig h e r
protein concentrations the activity decreases, probably because the substrate becom es the lim iting
factor.
°
^
>
c) K m-determination fo r mercaptopyruvate
The activity o f the m ercaptopyruvate sulfo tran s­
ferase is dependent on the concentration o f the su b ­
strate used, as show n in Fig. 3. F rom these d ata an
apparent K m for m ercaptopyruvate was d eterm in ed
to 2 m M , using crude Chlorella extracts. T hese d ata
were used to define the m ercap to p y ru v ate concen­
tration used in all further experim ents. T his concen­
tration was set to 5 m M ; hig h er concentrations in ­
creased the blanks, thus m aking this concentration a
com prom ise betw een optim al substrate co n cen tra­
tion and low blanks.
mg
protein
added
Fig. 2. Protein dependence of the mercaptopyruvate sulfo­
transferase activity from Chlorella. Conditions: As in
Fig. 1, however the pH was constant at 9.0 and the protein
concentration was varied as indicated.
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918
A. Schmidt • Occurrence of Mercaptopyruvate Sulfotransferase Activity in Photosynthetic Organisms
Mmol
mercaptopyruvate
added
ymol
DTE
added
Fig. 3. ^-determ ination for mercaptopyruvate. Condi­
tions: As in Fig. 1, however the pH was cept constant at 9.0
and mercaptopyruvate was varied as indicated.
Fig. 4. ^-determ ination for dithioerythritol. Conditions:
As in Fig. 1, however the pH was constant at 9.0 and the
dithioerythritol concentration was varied as indicated.
d) K m-determination for dithioerythritol
phototrophic bacterium (Rhodopseudom onas palus­
tris) were analyzed for m ercap to p y ru v ate su lfo tran s­
ferase activity using different thiol acceptors. T hese
data are sum m arized in T able I. F o r co m p ariso n we
have norm alized the data o b ta in ed w ith d ith io e ry ­
thritol as sulfur acceptor to 100% and calcu lated the
other data as percentage o f the d ith io ery th rito l
values. It is evident from these d ata th a t m e rc a p to ­
pyruvate sulfotransferase activity is d etected in each
species analyzed so far and th e d ith io ery th rito lcoupled rates are in the range o f (imol p y ru v ate
formed per mg proein and h o u r (Lem na: 6.51;
Spinacia: 2.11; Pisum: 1.77; Chlorella: 1.55; S yn ech o ­
coccus: 3.97; and Rhodopseudomonas: 5.56). In all
cases dithioerythritol is the best su lfu r acceptor; the
biological m onothiol g lu tath io n e is active too,
whereas cysteine supports low activity. S u lfite is an
acceptor for this reaction and we could d e m o n ­
strate the form ation o f th io su lfate (d ata n o t show n).
Note, that further addition o f m erc ap to p y ru v a te
enhanced this activity, d em o n stratin g th a t th is co m ­
pound is a donor and an acceptor for this reactio n .
Cyanide, thiocyanate, and th io su lfate are m o re or
less inactive.
A ddition o f d ithioerythritol to the assay system
enhanced the m ercaptopyruvate sulfotransferase
activity was shown in Fig. 4. T hese d ata do not
follow, however, sim ple M ichaelis-M enten kinetics,
since plotting o f these data according to L inew eaver
and Burk resulted in a broken line (insert o f Fig. 4)
indicating two different K m areas, one ab o u t
0.56 m M and one at ab o u t 5 m M . It suggests th a t
dithioerythritol could have a dual function leading
to an activation o f the enzym e besides its function
as an acceptor. F or ou r norm alized condition s a
concentration o f 10 m M d ith io ery th rito l was used,
since higher thiol concentrations have been found to
be inhibitory, if other thiol com pounds w ere used.
e) Distribution o f mercaptopyruvate
sulfotransferase activity and comparison
o f certain sulfur acceptors
Crude extracts from higher plants (Spinacia,
Lemna, Pisum), one green alga ( Chlorella fu sc a ),
one cyanobacterium (Synechococcus 6301), and one
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A. Schmidt • Occurrence of Mercaptopyruvate Sulfotransferase Activity in Photosynthetic Organisms
919
Table I. Acceptor specificity for mercaptopyruvate sulfotransferase.
Acceptor added
None
Dithioerythritol
Cysteine
Gluatathione
Mercaptoethanol
Mercaptopyruvate
Sulfite
Thiosulfate
Potassiumcyanide
Sodi umthiocyanate
Lemna
minor
10
100
7
22
34
19
14
Pisum
sativum
16
100
28
41
91
54
17
2
12
1
24
0.5
11
Spinacia
oleracea
19
100
19
41
51
36
15
3
9
14
Chlorella
fusca
8
100
13
28
39
31
13
9
14
11
Synechococcus
6301
1
100
10
37
52
28
27
3
14
16
R hodopseudomonas
palustris
13
100
8
43
80
51
75
13
27
16
Conditions: Each vessel contained (in nmol) in a total volume o f 1 ml: Tris-HCl pH 9.0: 100; mercaptopyruvate: 5;
acceptor: 10; and protein as indicated below. The reaction was stopped after 30 min at 37 °C and analyzed for pyruvate
formed. The following amounts of protein were added (|imols pyruvate formed using DTE = 100): Lemna: 0.79 mg
(2.57); Pisum 2.53 mg (2.24); Spinacia 1.32 mg (1.39); Chlorella 1.12 mg (0.879); Synechococcus 0.84 mg (1.70); Rhodo­
pseudomonas 0 . 8 mg (2 .2 2 ).
Discussion
Plants and algae are capable to d eg rad e cysteine
to inorganic sulfide and to oxidize it to sulfate as
discussed in the introduction ch a p te r leading to a
futile sulfur cycle [22], A nim als d egrade cysteine
either by oxygenation to cysteine sulfinic acid or
transaminate it to m ercaptopyruvate w hich is further
m etabolized to thiosulfate and pyruvate by a m e r­
captopyruvate sulfotransferase using sulfite as ac­
ceptor [23]. So far, evidence for the presence o f
m ercaptopyruvate sulfotransferase activity is m iss­
ing from plants and algae; W estley [23] stated in his
recent review th at this enzym e could be d etected
only in bacteria, fungi, and anim als.
A search for this enzyme activity was initiated by
our observation that Chlorella and spinach cells did
not contain a L-cysteine specific desulfhydrase
activity, suggesting that other mechanisms m ight be
involved in cysteine degradation. One possible
mechanism was demonstrated by the isolation of
D-cysteine specific desulfhydrases from Spinacia
and Chlorella [14, 15]; however, a racem ase con­
verting L-cysteine to D-cysteine was detected only by
indirect methods so far. Another possible pathw ay
for L-cysteine degradation would be com parable to
animal systems implying that m ercaptopyruvate is
an intermediate for cysteine catabolism. This would
be possible only, if a m ercaptopyruvate sulfotrans­
ferase activity is present in plants and algae, and
this had not been demonstrated so far [23]. The data
of this publication demonstrate for the first tim e the
presence of m ercaptopyruvate sulfotransferase activ­
ity in photosynthetic organism s.
T he catalytic properties o f th e plant type m e r­
captopyruvate sulfotransferase have b een d e te r­
m ined using Chlorella extracts. T h e final con d itio n s
to m easure this activity are a pH o f 9.0; a m e rc ap to ­
pyruvate concentration o f 5 m M and a d ith io ery thritol concentration of 10 m M . T he analysis o f
different thiol reagents clearly d em o n strated th a t
dithioerythritol is the best su lfu r acceptor lead in g to
pyruvate, oxidized d ith io ery th rito l and sulfide,
^ -d e te rm in a tio n s o f dith io ery th rito l lead to two
different A^m-values suggesting an ap p a ren t
of
0.56 and 5 m M . It is suggested th a t the enzym e can
be activated by thiols as observed for th e liver
enzyme [24]. Pyruvate was determ in ed using dinitrophenylhydrazin. D eterm in atio n o f sulfide using
the m ethylene-blue m ethod leads to erroneous re­
sults since m ercaptopyruvate is d eg rad ed nonenzymatic at acidic conditions yielding sulfide. It is
evident from th e d ata o f T ab le I th a t cyanide is less
effective com pared to d ith io ery th rito l; cyanide
probably reacts directly w ith m ercap to p y ru v ate
form ing a cyanohydrin ad d u ct thus q u en ch in g the
substrate [24]. M ercaptopyruvate sulfotransferase
from plants and algae used m ercap to eth an o l as an
acceptor; this clearly distinguished this reactio n
from thiosulfate reductase activities, w here m e r­
captoethanol is not a sulfur acceptor [25]. A d d itio n
o f sulfite leads to the form ation o f th io su lfate using
the m ethod o f Sörbo [26]; th erefo re this enzym e
activity could be active in m ercap to p y ru v ate degra-
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920
A. Schmidt • Occurrence of Mercaptopyruvate Sulfotransferase Activity in Photosynthetic Organisms
COOH
i
c=o
I
h2 c - s h
R -S H
0
II
CH3 -C-COOH
R -S -S H
R -S H
R -S -S -R
n s
dation to pyruvate and thiosulfate as suggested for
animals. Thiosulfate form ed w ould than be cleaved
by the thiosulfate reductase system [25] le ad in g to
sulfide and sulfite. It is clear th a t m e rc ap to p y ru v a te
itself is a donor and acceptor for this rea ctio n using
plant and algal systems; this was show n previously
for the bovine kidney system [21, 27], D T E w as p re ­
ferred as sulfur acceptor because the activity is
enhanced and the blanks are considerably low er d ue
to the thiol reducing capacity o f d ith io ery th rito l.
The evidence given in this pu b licatio n suggests
that m ercaptopyruvate could be an in te rm e d ia te in
cysteine degradation to sulfide and elem ental sulfu r
as suggested previously for Chlorella [28]. T he
following reactions sum m arized in Fig. 5 could be
involved in cysteine catabolism in plants.
M ercaptopyruvate could eith er be g en erated by a
transam inase reaction from cysteine and a su itab le
a-keto acid or by coupling to an am ino acid oxidase;
these reactions can be coupled principally e ith e r to
D - or L-cysteine. M ercaptopyruvate form ed is used
as a sulfur donor leading to thiosulfate if sulfite is
used as an acceptor or to a persulfide if a thiol is
used, with further reduction in both cases to free
sulfide. F orm ation o f sero valency sulfur can be
Fig. 5. Pathways for L- and D cysteine degradation in photo­
synthetic organisms.
explained by this system as well as suggested by
Krauss et al. [28] for Chlorella. T hus p ro d u ctio n o f
sulfide, am m onia, and pyruvate from cysteine could
be a coupled reaction n ot catalyzed by a cysteine
lyase. L-cysteine lyase activity was detected in
Acacia species [29, 30]; in tobacco [11], and in the
Cmciferae family [16], and we could detect L-cysteine
lyase activity in this fam ily as well. W e did n o t find
this activity how ever in spinach, L em na, Chlorella,
and Svnechococcus; therefore we were searching for
alternative possibilities. W e detected D-cysteine
specific lyase activities in plants and algae [14, 15],
however evidence for a racem ase converting lcysteine to D-cysteine is o b tain ed so far only by
indirect m ethods. Since in the D-cysteine lyase
reaction non-identified com ponents could be found
besides pyruvate and N H 4 (u n p u b lish ed ), we have
indicated this by the fo rm atio n as X as unknow n
product.
Clearly, different m echanism s for L-cysteine
catabolism have been suggested and could be rea l­
ized in plants and algae. T he precise d eterm in a tio n
o f the m echanism (s) o perative in vivo should be
analyzed studying the regulation o f the possible
pathways involved in cysteine degradation.
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A Schmidt • Occurrence of Mercaptopyruvate Sulfotransferase Activity in Photosynthetic Organisms
Acknowledgements
This work was sup p o rted by a grant from
D eutsche Forschungsgem einschaft. T he expert tech-
[1] A Schmidt, Encyclopedia Plant Physiol. VI, 4 8 1 -4 9 6
(1979).
[2] A. Schmidt and E. Krämer, Z. Naturforsch. 38 c,
446-450(1983).
[3] E. Krämer and A. Schmidt, Planta 160, 235-241
(1984).
[4] H. Rennenberg, Z. Pflanzenphysiol. 105, 3 1 -4 0
(1981).
[5] A Schmidt, I. Erdle, and H.-P. Köst, Z. Naturforsch.
37 c, 870-876(1982).
[6 ] C. Brunold and K H. Erismann, Experientia 30,
465-467 (1974).
[7] C. Brunold and K H. Erismann, Experientia 31,
508-509 (1975).
[8 ] I. K Smith, Plant Physiol. 5 5 ,3 0 3 -3 0 7 (1975).
[9] I. K Smith, Plant Physiol. 6 6 ,8 7 7 -8 8 3 (1980).
[10] A Schmidt, L. G. Wilson, J. Sekiya, and P. Filner,
Plant Physiol. 65, S-74 (1980).
[11] H. M. Harrington and I. K. Smith, Plant Physiol. 65,
151-155 (1980).
[12] J. Sekiay, A. Schmidt, H. Rennenberg, L. G. Wilson,
and P. Filner, Phytochemistry 21,2 1 6 3 -2 1 7 8 (1982).
[13] J. Sekiya, A Schmidt, L. G. Wilson, and P. Filner,
Plant Physiol. 70,4 3 0 -4 3 6 (1982).
[14] A Schmidt, Z. Pflanzenphysiol. 1 0 7,301-312 (1982).
[15] A. Schmidt and I. Erdle, Z. Naturforsch. 38 c,
428-435 (1983).
[16] D. I. Hall and I. K Smith, Plant Physiol. 72, 6 5 4 -6 5 8
(1983).
[17] C. Brunold and A. Schmidt, Planta 133, 8 5 -8 8
(1976).
921
nical assistance o f Karl M ayer is greatfully ack n o w l­
edged. We are indebted to Prof. Dr. K. K nobloch
for a generous gift o f Rhodopseudom onas palustris.
[18] A Schmidt, Z. Naturforsch. 27b, 183-192 (1972).
[19] A. Schmidt and U. Christen, Planta 140, 2 3 9 -2 4 4
(1978).
[20] M. Bradford, Anal. Biochem. 7 2 ,2 4 8 -2 5 4 (1976).
[21] R. Jarabak and J. Westley, Arch. Biochem. Biophys.
185,458-465 (1978).
[22] H. Rennenberg, H. Sekiya, J. Wilson, and P. Filner,
Planta 154,516-524(1982).
[23] J. Westley, in: Cyanide in Biology (B. Vennesland,
E. E. Conn, C. J. Knowles, J. Westley, and F. Wissing, eds.), Academic Press London, pp. 6 1 -7 6
(1981).
[24] E. Kun and D. W. Fanshier, Biochim. Biophys. Acta
3 3 ,2 6 -2 8 (1959).
[25] A Schmidt, I. Erdle, and B. Gamon, Planta, in press.
[26] B. H. Sörbo, Methods in Enzymol. II, 3 3 4 -3 3 7
(1955).
[27] R. Jarabak and J. Westley, Biochemistry 19, 9 0 0 -9 0 4
(1980).
[28] F. Krauss, W. Schäfer, and A. Schmidt, Plant Physiol.
74,176-182 (1984).
[29] M. Mazelis and R. K Creveling, Biochem. J. 147,
485-491 (1975).
[30] H. D. Gregor and R. Gmelin, Protoplasma 99,
117-123 (1979).
[31] M. Mazelis, N. Beimer, and R. K Creveling, Arch.
Biochem. Biophys. 120,371-378 (1967).
[32] N. F. Anderson and J. F. Thompson, Phytochemistry
18,1953-1958 (1979).
[33] M. Mazelis, K Scott, and D. Gallie, Phytochemistry
21,991-995 (1982).
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