From the
Rat liver mercaptopyruvate sulfurtransferase (MST) was purified
to homogeneity. MST is very similar to rhodanese in physicochemical
properties. Further, rhodanese cross-reacts with anti-MST antibody.
Both purified authentic MST and expressed rhodanese possess MST and
rhodanese activities, although the ratio of rhodanese to MST activity
is low in MST and high in rhodanese. In order to compare the active
site regions of MST and rhodanese, the primary structure of a possible
active site region of MST was determined. The sequence showed 66%
homology with that of rat liver rhodanese. An active site cysteine
residue (Cys
Mercaptopyruvate sulfurtransferase (MST, EC 2.8.1.2),
Amino acid sequencing was performed with a gas-phase
protein sequencer (ABI). Sequence homology was examined by means of
GENETYX (Software Development Co.).
A supernatant
of the rat liver homogenate after ultracentrifugation, purified MST,
and recombinant wild type rhodanese described below were loaded on a
SDS-polyacrylamide gel (10%) according to the method of Laemmli(1970).
Separated proteins were transferred to a nitrocellulose membrane
(Schleicher & Schuell) with an electrotransfer apparatus
(Sartorius) by the multiphasic buffer method according to Hirano
(1989). The first antibody was diluted (1:300) for use, and alkaline
phosphatase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch
Laboratories, Inc., 1:5,000) was used as a second antibody. Alkaline
phosphatase activity was detected with the usual color development
system.
To insert rhodanese cDNA into the pET-15b vector (Novagen) between
the NcoI and BamHI sites, each restriction site was
synthesized at sides of rhodanese cDNA by polymerase chain reaction
with pBs/Rho plasmid as a template. No mutation of polymerase chain
reaction product was confirmed by DNA sequencing with synthetic
oligonucleotide primers. Val was added to authentic rhodanese at the N
terminus in this study. This chimeric enzyme was designated wild type.
Dialyzed enzyme
was loaded onto a DE52 column (1.5
MST activity was
measured by the rate of pyruvate formation by a modification of the
method of Vachek and Wood(1972). The assay mixture contained 5 mM ammonium mercaptopyruvate, 25 mM mercaptoethanol, 0.1
mg/ml of bovine serum albumin, and 225 mM
2-amino-2-methyl-1,3-propanediol-HCl, and 5 µl of enzyme solution
in a final volume of 0.55 ml at pH 9.55. One unit of MST activity was
defined as 1 µmol of thiocyanate formed per minute.
SDS-PAGE during
purification of wild type and mutant rhodaneses is shown in Fig. 4. These expressed purified enzymes are also unstable and
can be stabilized in an ammonium sulfate solution. The amount of the
overexpressed enzymes obtained from 1 liter of culture varied from 3 to
7 mg. 54% of purified wild type rhodanese is recovered from the lysate.
The specific activity of the purified wild type enzyme shows 579.4
units/mg protein and about a 30-fold increase compared with that of the
lysate. SDS-PAGE shows that wild and mutant enzymes are 34.5-kDa
molecules (Fig. 4), which is in reasonable agreement with that
calculated from the deduced primary structure of rat liver rhodanese
(molecular weight, 33,176; Weiland and Dooley, (1991)). Purified
recombinant enzymes possess both rhodanese and MST activities. This is
not likely due to contamination by endogenous activities, because the
ammonium sulfate fraction (see ``Materials and Methods'') of
control E. coli contains endogenous rhodanese and MST
activities, 0.061 and 0.058 units/mg protein, respectively, which are
only 0.26 and 29.3% of rhodanese and MST activities, respectively, in E. coli that overexpresses wild type rhodanese. The total
endogenous activities are less than those of purified expressed
enzymes. Furthermore, we failed to recover the activities from the
control E. coli by the same purification procedures as
recombinant rhodanese.
Replacement of Arg
This mutagenesis study shows that
Arg
The present study showed that cytosolic MST
and mitochondrial rhodanese possess both MST and rhodanese activities,
although the ratio of rhodanese to MST activities was low in MST and
high in rhodanese. Further, these enzymes show striking similarity in
amino acid sequences and immunological cross-reactivity. In a
mutagenesis study, replacing two amino acids (Arg
All data except the ratio are shown as the mean
± standard error.
We thank Dr. Colin Thorpe of the University of
Delaware, Dr. Tomoko Nishino and Dr. Yoshihiro Amaya of Yokohama City
University, and Dr. Hiroyuki Hori of the Nippon Medical School for
helpful suggestions and discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
; site of formation of persulfide in
catalysis) and an arginine residue (Arg
; substrate
binding site) in rhodanese were also conserved in MST. On the other
hand, two other active site residues (Arg
and
Lys
) were replaced by Gly and Ser, respectively.
Conversion of rhodanese to MST was tried by site-directed mutagenesis.
After cloning of rat liver rhodanese, recombinant wild type and three
mutants (Arg
Gly and/or Lys
Ser) were constructed. The enzymes were expressed in Escherichia
coli strain BL21(DE3) with a T7 promoter system. The mutation of
these residues decreases rhodanese activity and increases MST activity.
(
)which catalyzes the transfer of sulfur ion from
mercaptopyruvate to mercaptoethanol, was first discovered in rat liver
(Meister, 1953; Wood and Fiedler, 1953; Meister et al., 1954;
Sörbo, 1954). This enzyme is widely distributed both in
prokaryotes and eukaryotes and is located mainly in the cytosol of
eukaryotic cells (Wood and Fiedler, 1953; Meister et al.,
1954; Kun and Fanshier, 1959; Jarabak and Westley, 1978). MST has been
proposed to play a role in cysteine degradation (Meister et
al., 1954) or in the biosynthesis of thiosulfate (Fasth &
Sörbo, 1973). Because eukaryotic MST has not been purified, its
molecular properties are unknown. Rhodanese (thiosulfate
sulfurtransferase, EC 2.8.1.1) is better understood. It was first found
in rat liver (Lang, 1933) and catalyzes the transfer of sulfur ion from
thiosulfate to potassium cyanide. Bovine liver rhodanese was isolated
and crystallized (Sörbo, 1953; Horowitz and DeToma, 1970), and the
rat liver enzyme has also been purified (Wasylewski et al.,
1979). Rhodanese exists as a monomer or dimer of identical subunits
(Volini et al., 1967) and is widely distributed in prokaryote
and eukaryote mitochondria (Ludwig and Chanutin, 1950; Sörbo,
1951; Duve et al., 1955). Primary structures of bovine liver
(Russell et al., 1978) and adrenal (Miller et al.,
1991), chicken liver (Kohanski and Heinrikson, 1990), human liver
(Pallini et al., 1991), and rat liver (Weiland and Dooley,
1991) rhodanese were determined from protein or deduced from cDNA. The
molecular mass of these enzymes is about 33 kDa. The crystal structure
of bovine liver rhodanese has been reported (Ploegman et al.,
1978a, 1978b, 1979), and its reaction mechanism has been clarified
(Westley and Nakamoto, 1962; Wang and Volini, 1968; Westley and Heyse,
1971; Schlesinger and Westley, 1974). Recombinant bovine liver and
adrenal rhodanese enzymes were overexpressed in Escherichia coli (Miller et al., 1991, 1992). Several biological functions
of rhodanese have been postulated: detoxification of cyanide (Lang,
1933), prevention of the formation of inorganic sulfide (Koj and
Frendo, 1962), and incorporation of sulfur into iron-sulfur protein
(Finazzi et al., 1971). Although rhodanese and MST were
believed to be different enzymes, Nishino et al.(1983, 1985)
reported that rhodanese or MST could transfer a sulfur ion from
thiosulfate or mercaptopyruvate to the molybdenum ligand of xanthine
oxidase, showing that these enzymes could catalyze similar reactions.
This paper compares the catalytic and structural properties of
cytosolic MST and mitochondrial rhodanese and shows that these enzymes
are evolutionarily related.
Chemicals
Mercaptopyruvate was synthesized
essentially by the method of Kun(1957). The amount of thiol group in
mercaptopyruvate was confirmed to be nearly one:one molar by titration
of thiol group with 5,5`-dithiobis(2-nitrobenzoic acid). Other
chemicals were analytical grade.
MST Purification
Liver MST was isolated from
7-week-old male Wistar rats. The rats were anesthetized with ether, and
their livers were excised. 200 g of livers were cut into small pieces
and homogenized with a Polytron (KINEMATICA) for 3 min on ice in 600 ml
of 5 mM potassium phosphate buffer containing 0.2 mM EDTA, pH 7.4. The homogenate was centrifuged at 130,000 g for 1 h at 4 °C. The 35-60% ammonium sulfate
precipitate was collected and dissolved in a minimal volume of 10
mM Tris-HCl buffer containing 0.2 mM EDTA, pH 7.8
(buffer A). After repeating this procedure with a second batch of
liver, the combined solution (obtained from 400 g of liver) was
dialyzed against three changes of 5 liters of the same buffer at 4
°C. The dialyzed enzyme was loaded onto a Q-Sepharose (Pharmacia
Biotech Inc.) column (3
24 cm) equilibrated with the same
buffer. The column was washed with 340 ml of the same buffer and then
with 600 ml of 60 mM Tris-HCl buffer containing 0.2 mM EDTA, pH 7.8. The enzyme was eluted with linear gradient of
60-500 mM Tris-HCl containing 0.2 mM EDTA, pH
7.8. MST-containing fractions were collected, and ammonium sulfate was
added to 60% saturation. The precipitate was dissolved in a minimal
volume of buffer A (final volume, 75 ml), dialyzed against three
changes of 2 liters of the same buffer at 4 °C, and loaded onto a
DEAE-cellulose (Whatman DE52) column (3
16.5 cm) equilibrated
with the same buffer. The column was washed with 420 ml of buffer and
then with 580 ml of 50 mM Tris-HCl buffer containing 0.2
mM EDTA, pH 7.8. The enzyme was eluted with a linear gradient
of 50-200 mM Tris-HCl containing 0.2 mM EDTA,
pH 7.8. The enzyme-containing fractions were collected, and ammonium
sulfate was added to 60% saturation. The precipitate was dissolved in
the minimal volume of 5 mM potassium phosphate buffer
containing 0.2 mM EDTA, pH 6.5 (final volume, 7 ml). The
enzyme solution was then applied to a G25-Sephadex (Pharmacia Biotech
Inc.) column (3
16.5 cm) equilibrated with the same buffer. The
enzyme-containing fractions were collected and loaded onto a
hydroxylapatite (Bio-Gel HTP, Bio-Rad) column (2.4
13 cm)
equilibrated with the same buffer. The column was washed with 180 ml of
buffer and developed with a linear gradient of 400 ml of buffer to 400
ml of 25 mM potassium phosphate buffer containing 0.2 mM EDTA and 0.1 M ammonium sulfate, pH 7.4. The
enzyme-containing fractions were collected, and ammonium sulfate was
added to 65% saturation. The precipitate was dissolved in a minimal
volume of 25 mM potassium phosphate buffer, pH 6.5 (final
volume, 1.5 ml) and concentrated with a Centricon 10 (final volume, 200
µl). The enzyme solution was desalted with Ampure
SA
(Amersham Corp.) and concentrated with a Centricon 10, (final volume,
320 µl). 160 µl of the solution was applied in two runs to a
HCA (hydroxylapatite for HPLC, Mitsui Toatsu Chemicals Inc.) column
(0.8
10 cm). The column was washed with 5 mM potassium
phosphate buffer, pH 6.5, for 5 min at a flow rate of 0.5 ml/min and
eluted with a 30-min linear gradient of the same buffer to 104 mM potassium phosphate buffer, pH 8. The enzyme-containing fractions
were collected and concentrated with a Centricon 10. 280 µl of the
concentrated enzyme solution was applied to two TSK (gel filtration for
HPLC, TOSOH Corp.) columns, which were connected in a series (3,000 SW,
2.15
60 cm and 2,000 SW
, 0.6
40 cm) and
were equilibrated with 0.2 M potassium phosphate buffer
containing 10 mM ammonium sulfate, pH 7.4. The
enzyme-containing fractions were concentrated with a Centricon 10 and
frozen until use.
Molecular Mass Determination
Purified enzyme was
applied to TSK 3,000 SW and 2,000 SW columns that were
connected in a series equilibrated with 0.2 M potassium
phosphate buffer containing 10 mM ammonium sulfate, pH 7.4,
and eluted with the same buffer at a flow rate of 0.5 ml/min. The
column was calibrated with bovine serum albumin (67 kDa), ovalbumin (43
kDa),
-lactoglobulin (36 kDa), and carbonic anhydrase (28 kDa).
Partial Primary Structure of MST
30 nmol of the
carboxymethylcysteinyl MST was digested with 10 µg of endoprotease
Lys-C (Boehringer Mannheim) in 25 mM Tris-HCl buffer
containing 1 mM EDTA, pH 8.5, at 37 °C for 8.5 h. 3 nmol
of alkylated enzyme was digested with 20 pmol of V8 protease (Takara
Shuzo Co., Ltd.) in 50 mM ammonium bicarbonate buffer, pH 7.8,
at 37 °C for 8.5 h. The digested samples were applied to a
reverse-phase C18 column (Capcellpak, Shiseido) and separated with a
linear gradient of acetonitrile between 15 and 80% in 0.1%
trifluoroacetic acid solution. Rechromatography was also performed for
some fractions.
Antibody Preparation and Western Blotting
The
highly purified enzyme solution (1.5 mg of protein) was injected
subcutaneously into a rabbit 5 times over 2 months. The antiserum
obtained from the immunized rabbit was precipitated with ammonium
sulfate and purified by DEAE-cellulose chromatography.
cDNA Cloning of a Rat Liver Rhodanese and Construction of
Expression Vector of Wild Type Rhodanese
5` sense primer
(CAAAGCTTGTATGGTGGATGTTTCGTGTG; 328-348 bp of rat liver rhodanese
cDNA with HindIII restriction site) and 3` antisense primer
(TCGAATTCTGTCAGGAAGTTCATGAAGGG; 628-648 bp with EcoR I
site) were synthesized according to the DNA sequence of the rat liver
rhodanese. A 323-bp product was obtained by polymerase chain reaction
with rat liver single-stranded cDNA as a template. Polymerase chain
reaction was performed with AmpliTaq DNA polymerase (Perkin-Elmer). The
probe was labeled with [-
P]dCTP by using a
random-primed DNA labeling kit (Boehringer Mannheim). After 5.5
10
clones of rat liver
gt11 cDNA library (CLONTECH)
were screened by plaque hybridization, 15 positive clones were
obtained. A 970-bp clone contained the longest reading frame of
rhodanese cDNA in the positive clones, but it showed a deletion of 29
base pairs in the 5` region. On the other hand, the reading frame was
followed by 115 bp of 3`-untranslated sequence. The deleted
double-stranded cDNA in the 5` region was reconstructed with four
synthetic oligonucleotides (between the PstI and RsrII sites) and added to the PstI-SphI
sites at the 5` end. The reconstructed rhodanese cDNA was subcloned to
pBluescript (pBs) at the PstI/EcoRI site (pBs/Rho).
Site-directed Mutagenesis
Replacement of
Arg (Arg
in recombinant wild type) by Gly
(R248G) and/or Lys
(Lys
in recombinant wild
type) by Ser (K249S) was performed according to the method of
Kunkel(1985). A fragment of rhodanese cDNA between KpnI (from
559 bp) and BamHI site (to the last) was excised from the
pBs/Rho plasmid. The fragment was inserted to pBs between the same
restriction sites. In the present study for mutagenesis, three
mutagenic oligonucleotides, CGCCACATGCGGCAAAGGGGTCA,
CACATGCCGCAGTGGGGTCACTG, and CGCCACATGCGGCAGTGGGGTCACTG, were
synthesized for R248G, K249S, and double mutants, respectively
(underlined codons indicate mutagenized sites). The accuracy of
mutagenesis was checked by DNA sequencing of the mutagenized rhodanese
cDNA with synthetic oligonucleotide primers. Each mutagenic fragment (KpnI/BamHI) was replaced with a fragment between KpnI and BamHI sites of pET/Rho plasmid.
Expression and Purification of Wild Type and Mutant
Rhodanese
These constructs were transformed into E. coli strain BL21(DE3). Transformed cells were cultured in LB medium
containing 50 mg/ml ampicillin at 27 °C. At an absorbance of 0.8 at
600 nm, expression was induced by adding 1 mM isopropyl
-D-thiogalactopyranoside and by increasing the culture
temperature to 37 °C. After 3.5 h at 37 °C, cells were
harvested by centrifugation at 6,000
g for 5 min. Wild
type and mutant rhodaneses were purified by a major modification of the
method of Miller et al.(1992). After adjusting the pH to 5.4
with 2 M glycine sulfate, pH 2.5, the lysates were
centrifuged, and the supernatant was fractionated with ammonium sulfate
(35-65% saturation). The precipitate was dissolved in a minimal
volume of 10 mM Tris-HCl buffer, pH 7.8 (buffer B), (final
volume, 2 ml) and dialyzed overnight against 2 liters of the same
buffer containing 2 mM sodium thiosulfate.
17 cm) equilibrated with
buffer B. The enzyme was eluted with the same buffer. Enzyme fractions
were collected and concentrated with a FILTRON-10 and a Centricon 10 to
a final volume of 10 ml. The enzyme solution was dialyzed overnight
against 2 liters of 5 mM sodium acetate buffer containing 2
mM sodium thiosulfate, pH 5.0, at 4 °C. The enzyme was
then loaded onto a CM52 (Whatman) column (0.5
12.7 cm)
equilibrated with 5 mM sodium acetate, pH 5.0. The column was
washed with the same buffer, and the enzyme was eluted with a linear
gradient to 1.05 M sodium acetate. Enzyme-containing fractions
were collected, concentrated in 5 mM sodium acetate, pH 5.0,
and stored at -20 °C after the addition of one-third volume
of 100% saturated ammonium sulfate solution.
Enzyme Assays
Rhodanese activity was measured by
following the rate of thiocyanate formation by a modification of the
method of Sörbo(1953). The assay mixture contained 60 mM sodium thiosulfate, 60 mM potassium cyanide, 40
mM NaHPO
, and 5 µl of enzyme
solution in a final volume of 0.5 ml at pH 5.0. The mixture was
incubated for 10 min at 25 °C. One unit of the enzyme was defined
as 1 µmol of pyruvate formed per minute.
Protein Determination
The protein concentration
was determined with a Coomassie protein assay kit (Pierce) with
crystalline bovine serum albumin as the standard.
Purification of MST
MST is unstable during
purification, but it has been found that ammonium sulfate stabilizes
the enzyme. This allowed rat liver MST to be purified to homogeneity by
including 10 mM ammonium sulfate in the late steps of the
purification procedure. A representative purification is summarized in . The specific activity of the purified enzyme shows a more
than 2,000-fold increase compared with that of a rat liver supernatant.
SDS-PAGE indicated that MST is composed of a 34-kDa subunit (Fig. 1). Gel filtration shows that the enzyme elutes as two
peaks having apparent molecular masses of 34.5 kDa and 53.5 kDa (data
not shown). The larger molecular weight fraction was converted to the
smaller one by treatment with 5 mM dithiothreitol in 0.2 M potassium phosphate buffer, pH 7.4, containing 10 mM ammonium sulfate (data not shown). Because both fractions from gel
filtration were identical on SDS-PAGE only after reduction with
dithiothreitol (data not shown), it was suggested that MST exists in
monomer and homodimer forms and that dimer formation involves an
intersubunit disulfide bond. Because the calculated molecular mass of
rat liver rhodanese is 33,176 kDa (Weiland and Dooley, 1991) and bovine
liver rhodanese was reported to exist in monomer-dimer equilibrium
mediated via disulfide bond formation (Volini et al., 1967),
MST seems to have similar physicochemical properties to rhodanese.
Figure 1:
SDS-PAGE of purified rat liver MST. MST
shows a subunit molecular mass of 34 kDa. Lane 1, 3 µg of
MST; lane 2, 6 µg of MST; M, molecular mass
markers.
Reactivity of Rhodanese with Anti-MST Antibody
In
order to know the immunological similarity between rat liver MST and
rhodanese, Western blot analyses were performed after SDS-PAGE of the
purified rhodanese and MST, as shown in Fig. 2. It was found that
anti-MST polyclonal antibody cross-reacts weakly with recombinant wild
type rat liver rhodanese (Fig. 2, lane 1). These results
suggest that there is a similarity between MST and rhodanese in protein
structure. In the soluble fraction of the rat liver homogenate (Fig. 2, lane 3), the main band and an additional faint
band were observed. The fact that the main band has a molecular weight
corresponding to MST suggested that it was MST. Another minor band
shows a larger molecular weight than that of MST but is slightly
smaller than that of recombinant liver rhodanese. It is possible that
this minor band is either mature rhodanese that leaked from
mitochondria during homogenization or another cross-reacting protein.
Figure 2:
Western blotting with anti-MST antigen. Lane 1, 1 µg of recombinant wild type rhodanese; lane
2, 0.28 µg of purified rat liver MST; lane 3, 16.4
µg of supernatant of the rat liver.
Partial Primary Structure of MST around the Active Site
and Comparison with the Sequences of Rhodanese
In order to
elucidate the structural difference between the active sites of MST and
rhodanese, a partial amino acid sequence of the rat liver MST was
determined by protein sequencing. Although the N terminus of MST is
blocked, 204-amino-acid sequences of internal peptides were determined
after proteolytic cleavage of the protein and purification of the
peptides (see ``Materials and Methods''). In a comparison of
the obtained sequence of MST with that of rat liver rhodanese, which
was deduced from the cDNA sequence (Weiland and Dooley, 1991), it was
found that the sequences of rat liver MST and rhodanese are highly
homologous and that the obtained sequence of MST covered the sequence
corresponding to that of the active site region of rhodanese (from
Gln to Pro
). The active site structure of
rhodanese was well characterized at the tertial structure level by
x-ray crystallographic studies with bovine liver rhodanese (Ploegman et al., 1978a, 1978b, 1979). The sequence of MST covering the
sequences around the active site of rhodanese are shown in Fig. 3in comparison with the sequences of rhodanese from various
sources. The degrees of sequence identity in these regions were 66.0%,
63.9%, 68.1%, and 87.5% between rat MST and rat, bovine, chicken, and
human rhodanese, respectively. In this part, one amino acid of rat MST
is deleted at the position corresponding to Val
of rat
rhodanese. The remaining part of the obtained sequence of MST has 57%
identity with the corresponding part of the sequence of rat rhodanese
(data not shown). These findings on the striking similarity between MST
and rhodanese in the amino acid sequence suggest that MST is
evolutionarily related to the enzyme family of rhodanese.
Figure 3:
Comparison of partial primary structure of
rat liver MST with sequences around active site of rhodaneses of rat,
bovine, chicken, and human liver. LEP, fragments obtained from
digestion of authentic rat liver MST with lysine endopeptidase; V8, fragments obtained from digestion of authentic rat liver
MST with V8 peptidase; Rhodanese-r, deduced primary structure
of rat liver rhodanese (Weiland and Dooley, 1991); Rhodanese-b, primary structure of purified bovine liver
rhodanese (Russell et al., 1978); Rhodanese-c, amino
acid sequence from purified chicken liver rhodanese (Kohanski and
Heinrikson, 1990); Rhodanese-h, deduced primary structure of
human liver rhodanese (Pallini et al., 1991); shaded
regions, identical amino acid residues; #1, arginine 185; #2, cysteine 246; #3, arginine 247; #4,
lysine 248 in authentic rat liver rhodanese.
It is
noteworthy that some catalytically important amino acid residues of
rhodanese were conserved among all rhodaneses and even MST, whereas
some residues were replaced by other amino acids in MST. Arg of rat rhodanese (Fig. 3, #1) is conserved in all
of these sequences. This residue is located at the entrance of the
pocket of the active center and substrate binding site in bovine
rhodanese (Ploegman et al., 1978a, 1978b, 1979). Cys
of rat rhodanese (Fig. 3, #2) is also conserved
among all these sequences. This residue is identified to form the
persulfide and transfer sulfur ion between substrates in bovine
rhodanese (Ploegman et al., 1978a, 1978b, 1979; Weng et
al., 1978). On the other hand, Arg
of rat rhodanese (Fig. 3, #3) is conserved in bovine and chicken
rhodaneses but is replaced with a Gly in MST and human rhodanese.
Lys
of rat rhodanese (Fig. 3, #4) is also
conserved in bovine and chicken rhodaneses but is replaced with a Ser
in MST and human rhodanese. This residue is located at the entrance of
the pocket of the active center and substrate binding site in bovine
rhodanese (Ploegman et al., 1978a, 1978b, 1979). It should be
noted that the primary structure of human rhodanese deduced from cDNA
(Pallini et al., 1991) is very similar to that of rat liver
MST and the four amino acid residues described above are identical with
those in MST. As the purified MST has a weak rhodanese activity, as
mentioned below, it is possible that the reported human rhodanese might
in fact be a MST.
Expression and Purification of Wild Type and Mutant Rat
Liver Rhodanese
We found that rhodanese and MST possess both
rhodanese and MST activities, but their ratios are different. The ratio
of the activity of rhodanese to MST is high in rhodanese and is low in
MST as shown in I. To exclude the possibility of
contamination of enzymes by each other, to elucidate the function of
the two amino acid residues in rhodanese and MST activities
(Arg
Gly and Lys
Ser)
discussed above (Fig. 3), and to attempt to convert rhodanese to
MST, we cloned rat liver rhodanese cDNA and constructed three
recombinant mutants (see ``Materials and Methods'' and ). We confirmed that the nucleotide sequence was identical
to that reported previously by Weiland and Dooley(1991). The wild type
and three mutant rhodanese cDNAs were identified as described under
``Materials and Methods.'' Protein sequencing showed that the
amino acid sequences in the N terminus of all of the expressed
rhodaneses agreed with the cDNA sequences. All are preceded by Val (not
Met) as expected from construction of the expression system described
under ``Materials and Methods.'' These findings also confirm
that the proteins obtained are recombinant rhodaneses and not authentic
enzymes that possibly exist in E. coli.
Figure 4:
SDS-PAGE of purification steps of the
wild type rhodanese and purified mutant rhodaneses. M,
molecular mass marker. Lanes 1-5 show the purification
step of wild type rhodanese. Lane 1, lysate; lane 2,
supernatant after acid precipitation; lane 3, ammonium sulfate
fraction (35-65%); lane 4, DE52 fraction; lane
5, CM52 fraction; lane 6, purified K249S enzyme; lane
7, purified R248G enzyme; lane 8, purified double mutant
enzyme.
Effect of Mutagenesis and Kinetic Properties of
Enzymes
In the study of bovine liver rhodanese (Westley and
Heyse, 1971; Schlesinger and Westley, 1974), the reaction mechanism was
reported to follow a ping-pong kinetic pattern. But this study showed
that the double reciprocal plots of velocity versus KCN
concentration did not show a straight line when rhodanese activities
were measured with the wild type and mutant rhodaneses and the purified
rat liver MST (data not shown). Apparent K values for thiosulfate were therefore determined with a
constant concentration of potassium cyanide at 60 mM in this
study. On the other hand, the double reciprocal plots of all these
rhodaneses and the purified rat liver MST show a sequential kinetic
pattern when MST activity is measured (data not shown), as in the
partially purified bovine kidney MST (Jarabak and Westley, 1978, 1980).
(corresponding to Arg
in authentic rat rhodanese) with Gly in recombinant rhodanese
(R248G in ) does not affect rhodanese activity; K
and k
for
thiosulfate are not essentially affected (I), suggesting
that Arg
is not critical for rhodanese activity. On the
other hand, this replacement increases MST activity; k
for MST activity and k
/K
are increased
about 35-fold and about 30-fold of that in wild type, respectively,
without significant change in K
for
mercaptopyruvate (I). Replacement of Lys
(corresponding to Lys
in authentic rat rhodanese)
with Ser in recombinant rhodanese (K249S in ) decreases
rhodanese activity, K
for thiosulfate is
increased to about 12 times that in wild type (I), and
both k
and k
/K
using
thiosulfate are decreased to about one-fifth and about one-sixtieth of
that in wild type, respectively (I). On the other hand,
this replacement does not essentially affect MST activity; K
and k
for
mercaptopyruvate are not affected (I). Double replacement
of Arg
and Lys
with Gly and Ser,
respectively, in rhodanese () not only decreases rhodanese
activity but also increases MST activity as shown in I.
The ratio of the apparent k
for rhodanese
activity/apparent K
for thiosulfate to
the k
for MST activity/K
for mercaptopyruvate is the largest in mutant rhodaneses
(about 340 times that in wild type) (I). These data
indicate that the double mutagenesis is most effective in the
conversion of rhodanese to MST.
and Lys
are critical residues in
determining the rhodanese/MST activity ratio of this family enzyme. In
this study, replacement of Lys
with Ser decreased
rhodanese activity. Another report showed that the replacement of this
Lys with Ala of bovine liver rhodanese completely eliminated rhodanese
activity (Luo and Horowitz, 1994). It is possible that the replacement
of Lys
by an alanine residue changes the conformation
around the active site to a higher degree than replacement by a serine
residue. The facts that the structure of MST is similar to that of
rhodanese in the active site region and that they both catalyze
sulfurtransferase reactions suggest that the two enzymes should have
some similarity in the reaction mechanism. The cysteine residue in the
rhodanese is known to be involved in catalysis as a residue responsible
for persulfide formation in the thiosulfate sulfurtransferase reaction
(Ploegman et al., 1979). Because this residue is also
conserved in MST, it is not surprising that MST can catalyze the
thiosulfate sulfurtransferase reaction. This cysteine residue might
also be considered to play a role for persulfide formation between
protein and mercaptopyruvate during catalysis. However, the facts that
the residue of Lys
, which was considered to be important
for thiosulfate binding, is replaced by Ser in MST and that the
site-directed mutagenesis of this residue of rhodanese decreased k
values without dramatic change of K
for thiosulfate suggest that the
electrostatic interaction between the minus charge of the oxygen atom
of thiosulfate and the plus charge of lysine residue might be important
for transferring outer sulfur atom of thiosulfate to form persulfide
intermediate. On the other hand, in mercaptopyruvate sulfurtransferase
reaction, the existence of a plus charge seems to disturb the formation
of persulfide. However, the mutants of rhodanese do not possess the
same degree of MST activity as the purified rat liver enzyme,
suggesting that a factor(s) other than the residues mutated in this
experiment may be involved in the higher catalytic activity in the
authentic MST enzyme.
and
Lys
by Gly and Ser, respectively) converted rhodanese to
MST most effectively. These findings strongly suggested that the two
enzymes are evolutionarily related members of the same
``family''. Although rhodanese is well characterized in its
properties and the enzyme is known to localize in mitochondria (Ludwig
and Chanutin, 1950; Sörbo, 1951; Duve et al., 1955), the
physiological role of the enzyme is not well understood. On the other
hand, MST is less characterized than rhodanese, although the enzyme was
discovered quite a long time ago (Meister, 1953) and is known to
localize in the cytosolic fraction (Wood and Fiedler, 1953; Meister et al., 1954; Kun and Fanshier, 1959; Jarabak and Westley,
1978). This study provides new insights related to the investigation of
the physiological roles of these enzymes and the mechanism of
distribution of these enzymes in two different compartments of the
cell.
Table: Purification of MST from rat liver
Table: Amino acid residues in recombinant
rhodaneses and MST
Table: Kinetic properties of wild type and mutant
rhodaneses and MST
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.