(Received for publication, February 22, 1995; and in revised form, March 24, 1995 )
From the
Glutathionylspermidine (GSP) synthetases of Trypanosomatidae and Escherichia coli couple hydrolysis of ATP (to ADP and
P) with formation of an amide bond between spermidine
(N-(3-aminopropyl)-1,4-diaminobutane) and the glycine
carboxylate of glutathione (
-Glu-Cys-Gly). In the pathogenic
trypanosomatids, this reaction is the penultimate step in the
biosynthesis of the antioxidant metabolite, trypanothione
(N
,N
-bis(glutathionyl)spermidine),
and is a target for drug design. In this study, GSP synthetase was
purified to near homogeneity from E. coli B, the gene encoding
it was isolated and sequenced, the enzyme was overexpressed and
purified in quantity, and the recombinant enzyme was characterized. The
70-kDa protein was found to have an unexpected second catalytic
activity, glutathionylspermidine amide bond hydrolysis. Thus, the
bifunctional GSP synthetase/amidase catalyzes opposing amide
bond-forming and -cleaving reactions, with net hydrolysis of ATP. The
synthetase activity is selectively abrogated by proteolytic cleavage 81
residues from the C terminus, suggesting that the two activities reside
in distinct domains (N-terminal amidase and C-terminal synthetase).
Proteolysis at this site is facile in the absence of substrates, but is
inhibited in the presence of ATP, glutathione, and
Mg
.
A series of analogs was used to probe the
spermidine-binding site of the synthetase activity. The activity of
diaminopropane as a substrate, inactivity of the C-C
diaminoalkanes, and greater los
s of specificity for analogs
modified in the 3-aminopropyl moiety than for those modified in the
4-aminobutyl moiety indicate that the enzyme recognizes predominantly
the diaminopropane portion of spermidine and corroborate N-1 (the
aminopropyl N) as the site of glutathione linkage (Tabor, H. and Tabor,
C. W.(1975) J. Biol. Chem. 250, 2648-2654). Trends in
K
and k
for a set
of difluoro-substituted spermidine derivatives suggest that the enzyme
may bind the minor, deprotonated form of the amine nucleophile.
The tripeptide, glutathione (-Glu-Cys-Gly, GSH), and the
polyamine, spermidine
(N-3-aminopropyl-1,4-diaminobutane), are present at high
concentrations (0.1-10 mM) in both eukaryotic and
bacterial cells (see, for example, Refs. 1, 2 for reviews). Among the
many functions of GSH is detoxification of xenobiotics and reactive
oxygen species, which it serves by virtue of its nucleophilic and
redox-active cysteine sulfhydryl group. As an antioxidant, GSH quenches
oxygen radicals (O, HO) by direct reduction and acts as a cofactor in
the scavenging of peroxides via the GSH peroxidase-GSH reductase
couple. The polyamines have also been ascribed a variety of roles,
including charge neutralization and complexation of the anionic
phosphodiester backbone of DNA. An intersection of GSH and spermidine
metabolism, in the form of the amide-linked conjugate,
N
-glutathionylspermidine (see for
structure), was first observed in Escherichia coli(3, 4) but has not been characterized with regard to its
physiological significance. More recently, it was shown that
glutathionylspermidine and the corresponding bis-amide,
N
,N
-bis(glutathionyl)spermidine
(trivial name trypanothione, see for structure), are
present in the pathogenic protozoa of genera Trypanosoma and
Leishmania(5, 6) . Several observations suggest
that the GSH-spermidine conjugates are physiologically important to
these organisms: first, the parasites appear to lack typical catalase
and peroxidase hemoproteins
(7, 8) , enzymes which
function in oxidant defense in other organisms; second, a considerable
portion of their GSH (a critical antioxidant) is conjugated with
spermidine
(6) ; and third, they lack the typical glutathione
peroxidase-glutathione reductase enzyme couple
(9) , but instead
utilize an analogous system based on
trypanothione
(10, 11, 12) . On the basis of
these observations, it has been suggested that the parasites are
dependent on trypanothione metabolism for oxidant defense, and, in view
of their inherent sensitivity to compounds which can induce oxidative
stress (i.e. redox cyclers), that trypanothione metabolism
might be a fruitful target for new antiparasitic drugs
(5) .
Indeed, several existing drugs (e.g. organic arsenicals,
nitrofurans, and difluoromethylornithine) have been proposed to exert
their trypanocidal activity by interfering with some aspect of
trypanothione metabolism (5, 13-16).
Earlier work in our
laboratory was directed toward molecular characterization of
trypanothione reductase and identified this enzyme as a new target for
antitrypanosomal drug
design
(10, 17, 18, 19, 20, 21) .
More recently, we have turned our attention to the ATP-hydrolyzing,
amide-bond-forming (ATP-amide) enzymes, glutathionylspermidine
synthetase (GSP(
)
synthetase), and trypanothione
synthetase (T(SH)
synthetase), which catalyze the
biosynthesis of trypanothione from glutathione and spermidine
()
(22, 23) . We purified both synthetases
to homogeneity in minute quantities from the insect trypanosomatid
Crithidia fasciculata(23) , but to date have not
succeeded in isolating the genes that encode them. As noted above, the
intermediate in trypanothione synthesis, glutathionylspermidine, was
first identified in E. coli(3, 4) , and a GSP
synthetase activity was partially purified
(24, 25) .
With the aim of understanding in detail the () bacterial
and parasitic enzymes that conjugate GSH and spermidine, we have now
purified E. coli GSP synthetase to near homogeneity, isolated
and determined the sequence of the gene which encodes it, overproduced
it in quantities sufficient for characterization, and used a series of
analogs to examine features of the spermidine binding site. In
addition, we have shown that the 70-kDa protein surprisingly possesses
an opposing GSP amidase activity and that the two reactions are most
likely catalyzed at distinct active sites.
[S]Glutathionylspermidine was prepared
enzymatically from [
S]GSH by using pure,
recombinant GSP synthetase. The reaction contained in a final volume of
1.76 ml: 39 µM [
S]GSH (1.5
Ci/mmol), 2.0 mM spermidine, 2.0 mM ATP, 2.6
mM MgCl
, 5.1 mM dithiothreitol, 41
mM Na-PIPES buffer, pH 6.8, and 3.1 µM GSP
synthetase. The reaction was allowed to proceed at 37 °C for 11 min
and was quenched by a 2.5-min incubation at 90 °C. The solution was
centrifuged briefly to pellet denatured protein and was loaded onto a
1.5-ml SP-Sepharose (Pharmacia) column (NH
counter ion) equilibrated in H
O. The column was
washed with 2
1 ml aliquots of 40 mM Na-PIPES, pH 6.8,
containing 4 mM EDTA and 4 mM dithiothreitol (to wash
through [
S]GSH, which does not bind to the
column, and to elute bound Mg
), followed by 3
1 ml of 150 mM ammonium acetate, pH 5.2.
Glutathionylspermidine was then eluted with 4
1 ml of 300
mM ammonium acetate, pH 5.2 (spermidine elutes at >500
mM ammonium acetate). Fractions 2 and 3 of the 300 mM
ammonium acetate wash, which contained >98% of the
glutathionylspermidine and 79% of the total radioactivity were
evaporated to dryness in vacuo. The material was redissolved
in 250 µl of 10 mM dithiothreitol. Analysis by thin layer
electrophoresis on a cellulose plate (as described below for the
radiometric GSP synthetase assay) revealed that >90% of the isolated
radioactivity co-migrated with authentic glutathionylspermidine, and
could be converted by treatment with GSP synthetase/amidase to a
species which co-migrated with GSH.
All steps in the purification were performed at 4 °C.
All buffers contained (in addition to the buffering component) 1
mM EDTA and 5 mM dithiothreitol, and the pH of each
was adjusted at room temperature. In the most successful purification,
20 g of cell paste was resuspended in 55 ml of 50 mm Na-HEPES, pH 7.5
(buffer A). Phenylmethylsulfonyl fluoride, benzamidine, and trypsin
inhibitor were added to concentrations of 500 µM, 6
mM, and 0.04 mg/ml, respectively. The cells were lysed by
passage through a French pressure cell at 14,000-16,000
pounds/square inch. The lysate was centrifuged at 17,000 g for 20 min, and the supernatant was brought to 1% (w/v) in
streptomycin sulfate by dropwise addition of a 6% solution in buffer A
with stirring. The solution was centrifuged at 17,000
g for 20 min, and the supernatant was brought to 33% of saturation
in ammonium sulfate by addition of the solid (0.18 g/ml) with stirring.
This solution was centrifuged at 17,000
g for 20 min,
and the supernatant was brought to 65% of saturation in ammonium
sulfate by addition of the solid (0.17 g/ml) with stirring. The sample
was centrifuged at 17,000
g for 20 min, and the pellet
was redissolved in 10 ml of buffer A. This solution was centrifuged
briefly at 17,000
g to pellet undissolved material and
then was desalted through a 2.6
26-cm Sephadex G-25 column
equilibrated in 50 mM Bis-Tris propane-HCl, pH 7.15 (buffer
B). The fractions containing protein (116 ml) were pooled and loaded
onto a 2.6
20-cm DEAE-Sepharose column equilibrated in buffer
B. The column was developed with a 50-ml gradient of 0-160
mM NaCl in buffer B, then with a 300-ml gradient of
160-400 mM NaCl in buffer B. Fractions containing GSP
synthetase activity (47 ml eluting at
170-210 mM
NaCl) were pooled. The pool was dialyzed against buffer B, then loaded
onto a mono-Q HR 10/10 column (Pharmacia), equilibrated in buffer B.
The column was developed with a 20-ml linear gradient of 0-180
mM NaCl in buffer B, then with a 100-ml linear gradient of
180-360 mM NaCl in buffer B, and fractions containing
enzyme activity (10 ml eluting at
150-190 mM NaCl)
were pooled. At this stage, an attempt was made to bind the enzyme to
Mono-S resin, as follows. The Mono-Q pool was dialyzed against 25
mM Na-MES buffer, pH 6.35 (buffer C). The pool was then
injected onto a Mono-S HR 10/10 column (Pharmacia) equilibrated in
buffer C, and the column was washed with buffer C prior to development
with a NaCl gradient. All enzyme activity was found in the wash
fractions, and these were pooled and loaded onto a 2.6
12-cm
hydroxylapatite column equilibrated in 20 mM potassium
phosphate buffer, pH 6.8 (buffer D). The column was developed with a
20-ml gradient of 20-68 mM potassium P
, then
with a 300-ml gradient of 68-212 mM potassium
P
. Fractions containing activity (60 ml eluting at
130-160 mM P
) were pooled, and the pool was
concentrated to 15 ml in a Centriprep 30 concentrator (Amicon). The
pool was diluted with H
O to 30 ml, and 5.2 g of solid
ammonium sulfate was added over several minutes with stirring. This
solution was centrifuged at 2,000
g for 10 min and
then loaded onto a phenyl-Superose HR 10/10 column (Pharmacia)
equilibrated in buffer D containing 1.2 M ammonium sulfate.
The column was developed with a 30-ml linear gradient of 1.2-0.78
M ammonium sulfate in buffer D, then with a 100-ml linear
gradient of 0.78-0.36 M ammonium sulfate in buffer D.
Fractions containing activity (9 ml eluting at 0.64-0.60
M ammonium sulfate) were pooled and dialyzed against buffer C.
This solution was loaded onto a Mono-S HR 5/5 column, which was
developed with a 2-ml linear gradient of 0-100 mM NaCl
in buffer C followed by a 40-ml linear gradient of 100-500
mM NaCl in buffer C. Fractions containing enzyme activity (5
ml eluting at 210-260 mM NaCl) were pooled, and the pool
was concentrated to 0.25 ml. This sample was analyzed by denaturing
polyacrylamide gel electrophoresis (SDS-PAGE) on a 7.5% gel, and the
gel was electroblotted to a polyvinylidene fluoride membrane. The two
bands detected by Ponceau S staining of the membrane (
70 and
90 kDa) were excised and submitted to the Harvard Microchemistry
Facility for endoproteinase lys C (which proteolyzes after Lys
residues) digestion and sequence determination of proteolytic peptides.
DNA from the Kohara -phage
clones
(26) was isolated from infected cultures of E. coli strain ZK126
(28) by using a kit from Qiagen and following
the manufacturer's instructions.
Purification of GSP synthetase from BL21DE3/pGSP cell paste was accomplished by a procedure modified from that used to purify the enzyme from E. coli B. Lysis of the cells and streptomycin sulfate and ammonium sulfate precipitation steps were carried out as described, except protease inhibitors (phenylmethylsulfonyl fluoride, benzamidine, and trypsin inhibitor) were not added to the lysis buffer. Desalting following ammonium sulfate precipitation was accomplished by dialysis against buffer B. DEAE-Sepharose chromatography was as described. Following this step, ammonium sulfate was added to the pool to a concentration of 1.2 M. This solution was loaded on the phenyl-Superose HR 10/10 column, which was developed as described. Fractions of highest specific activity were pooled. At this stage, GSP synthetase was estimated by SDS-PAGE to be 95% pure. When greater purity was desired, a portion of the phenyl-Superose pool was dialyzed against buffer B. This solution was loaded onto a Mono-Q HR 5/5 column (Pharmacia) equilibrated in buffer B. The column was developed with a 10-ml linear gradient of 0-100 mM NaCl in buffer B, then with a 45-ml gradient of 100-250 mM NaCl in buffer B. Fractions with highest specific activity were pooled.
To prove chromatographic
resolution of the native and trypsin-cleaved proteins, a solution
containing 0.5 mg of each was injected on the Mono-Q HR 5/5
column. The column was developed with a 5-ml linear gradient of
0-150 mM NaCl in buffer B, then with a 30-ml linear
gradient of 150-250 mM NaCl in buffer B. Fractions were
analyzed for protein concentration, amidase activity, and synthetase
activity (by the spectrophotometric assay) and were characterized by
SDS-PAGE.
Figure 1: Mono-S chromatography as final step in purification of GSP synthetase from E. coli B. A, absorbance at 280 nm (filled circles) and enzyme activity (open circles) of column fractions. B, SDS-PAGE analysis of pooled fractions 24-30.
Figure 2: Physical map of the region of the E. coli chromosome containing gsp (26).
Figure 3:
Sequences of gsp and the
predicted GSP synthetase/amidase protein. The dots above the
nucleotide sequence mark the positions to which the nucleotide and
amino acid numbering (to the left and right of the dots,
respectively) refer. Peptide sequences obtained from the 70-kDa
protein purified from E. coli B are
underlined.
A second partial open reading frame of 651 bases was
identified 300 bases downstream of gsp. The protein
sequence predicted is 85% identical over 217 amino acids with the
E. coli pit gene product, which is involved in low affinity
phosphate transport. As pit has been mapped to 77 min of the
E. coli chromosome
(33) , this sequence may represent a
second pit gene.
Figure 4: SDS-PAGE to monitor purification of recombinant GSP synthetase/amidase from overproducing strain BL21DE3/pGSP. Lanes 1 and 7, molecular weight markers; 2, total cell lysate; 3, lysate supernatant; 4, 1% streptomycin sulfate supernatant; 5, 33% saturation ammonium sulfate supernatant; 6, 66% saturation ammonium sulfate pellet after dialysis; 8, DEAE-Sepharose pool; 9, phenyl-Superose pool; 10, Mono Q pool.
A series of spermidine analogs, previously
used by Folk and co-workers (34) to probe the spermidine-binding site
of deoxyhypusine synthase, was tested with GSP synthetase
(Table II). In this series, either the 3-aminopropyl or the
4-aminobutyl substituent on the central amine (N-4) is modified.
Addition-subtraction of a methylene unit to/from the 4-aminobutyl
moiety (1 or 2) or conversion of the
-CH-NH
unit to a cyano- substitutent (4)
has a much smaller effect on the substrates' specificity constant
(k
/K
) than the
equivalent modification of the 3-aminopropyl moiety (compare 1-3 and 4-5). These results, especially the
observation that 4 is a substrate while 5 is completely
inactive, again corroborate N-1 as the site of acylation
(4) .
The effects on recognition and catalysis of perturbing the
pK values of the spermidine amine groups
were assessed by measuring catalytic parameters for 2,2- 6,6-, and
7,7-difluorospermidine derivatives (6-8), a series of
analogs previously used by the Merrell Dow group to probe the active
site of spermine synthase
(35) . As indicated in Table III,
difluoro-substitution drastically decreases the
pK
value of the nearest amine
group(s)
(35) . With GSP synthetase, the difluorospermidine
derivatives all have k
values identical with
that of spermidine. In contrast, K
is
markedly affected by difluoro-substitution. The modest (3-fold)
increase observed in 8 may reflect a binding preference for the
N-8-protonated species, of which the concentration in solution at pH
6.8 is reduced 2.4-fold by difluoro-substitution at position 7.
Likewise, the more than 20-fold increase in K
seen in 7 most likely reflects a binding preference for
the N-4-protonated species, of which the concentration is 13-fold lower
than for spermidine. The largest effect observed is for substitution at
position 2 (6), which improves (i.e. decreases)
K
so that it becomes immeasurable by the
coupled assay. In order to quantify this effect,
K
values for 6 and for spermidine
were determined in 50 mM Bis-Tris propane-HCl, pH 6.8. This
buffer contains a diaminopropane moiety and inhibits with respect to
spermidine, thereby increasing the apparent K
for 6 into the concentration range where it can be
accurately measured with the coupled assay. The
K
values for 6 and spermidine
determined in the presence of this buffer (Table III, in
parentheses) indicate that difluoro-substitution at position 2 improves
K
25-fold relative to spermidine.
Substitution at this position decreases the pK
values of both N-1 and N-4, and the effect of decreasing the N-4
pK
(as observed for 7) opposes the
net increase in specificity observed for 6. Thus, the data
suggest that the intrinsic N-1 effect is a large increase in
specificity, which in turn suggests that the enzyme may have a
preference for binding the N-1-deprotonated form of spermidine. For 6, this form is present at 330-fold higher concentration than
for spermidine. The aforementioned opposing N-4 effect may prevent the
preference for N-1-free amine binding from being fully expressed in 6. The proposal that GSP synthetase binds the N-1-deprotonated
form of spermidine is consistent with the inverse pH dependence of the
spermidine K
.
Figure 5: Qualitative thin layer electrophoresis and ninhydrin staining assay to demonstrate GSP amidase activity and its pH dependence. Assays were carried out in a mixed buffer system containing 25 mM each of Bicine, MES, Bis-Tris propane, and HEPES adjusted to the indicated pH with HCl or NaOH. They contained 6.2 mM glutathionylspermidine, 20 mM dithiothreitol, and 7 µM GSP synthetase/amidase. Analysis was carried as described under ``Experimental Procedures.''
Figure 6:
Trypsin proteolysis of native GSP
synthetase/amidase into a stable 60- kDa species devoid of
synthetase activity. A, time course of cleavage monitored by
SDS-PAGE. B, time course of loss of GSP synthetase
activity.
Figure 7: Mono-Q chromatography (as described under ``Experimental Procedures'') of native and trypsin-cleaved GSP synthetase/amidase. A, superimposed chromatograms from separate injections of species: protein concentration (filled symbols and solid lines) and GSP amidase activity (open symbols and dashed lines) for native protein (circles) and trypsin-cleaved protein (squares). The values for the trypsin-cleaved protein have been multiplied by 2.5 to normalize for the amount of protein injected. B, chromatogram from coinjection of species in 1:1 ratio: protein concentration (filled circles and dashed line), GSP synthetase activity (open circles and solid line) and GSP amidase activity (open squares and dotted line). The inset below the x axis shows SDS-PAGE analysis of the fractions.
Figure 8:
Test for protection against trypsin
cleavage of GSP synthetase by substrates and/or substrate analogs.
Proteolysis was carried at as described under ``Experimental
Procedures,'' with the indicated substrates and/or analogs
included. The designation - or + above the gel indicates the
absence or presence of 2.5 mM
MgCl.
As an initial step in molecular characterization of GSP
synthetase, the nature of the spermidine-binding site was probed. The
enzyme appears to recognize predominantly, though not exclusively, the
diaminopropane moiety of spermidine. The C diaminoalkane
itself is a substrate (albeit with elevated
K
), and various modifications of the
4-aminobutyl substituent are tolerated. Conversely, modifications of
the 3-aminopropyl substitutent abrogate activity (except in the case of
bis(4-aminobutyl)amine). These results are consistent with the
conclusion of Tabor and Tabor
(4) that glutathionylation occurs
predominantly at N-1, although the activity of bis(4-aminobutyl)amine
raises the possibility that modification of N-8 can also occur.
Interestingly, spermine
(N,N`-bis(3-aminopropyl)-1,4-diaminobutane) is a good
substrate (K
<20 mM,
k
6 s
), and the ratio of
moles of NADH consumed in the coupled assay/mole of spermine added
suggests that both aminopropyl substituents are glutathionylated.
Results with the difluorospermidine derivatives give insight into
the charge complementarity of the spermidine-binding site. Not
surprisingly, it appears that protonation of N-4 and N-8 is favored for
binding. In contrast, the simplest interpretation of the improved
K of 2,2-difluorospermidine is that the
enzyme has a preference for binding the N-1-deprotonated species. Given
the evidence that N-1 is the site of glutathionylation
(4) , this
observation is of mechanistic interest. If GSP synthetase is, as
expected, mechanistically similar to other ATP-amide enzymes such as
glutamine synthetase
(38, 39) , glutathione
synthetase
(40) , and D-ala:D-ala
ligase
(41) , N-1 acts as nucleophile to attack a
glutathionylphosphate intermediate (). One might expect
that the enzyme would possess an active site base to deprotonate the
ammonium form of N-1. In contrast to this expectation, the data suggest
that the enzyme selects from solution the minor N-1-free amine species.
This tentative conclusion is reminiscent of work on glutamine
synthetase
(42) and D-alanine:D-alanine ligase
(43), for which binding of the free amine nucleophile has also been
proposed. Thus, this catalytic strategy may prove to be a general
feature of ATP-amide enzymes ().
The proteolysis and
substrate protection experiments also provide some mechanistic insight.
In both D-ala:D-ala ligase
(37, 44) and glutathione
synthetase
(36, 45, 46) , a flexible -loop
provides a labile site for proteolysis in the unliganded
protein
(47) . Addition of the proper combination of substrates
and/or inhibitors partially protects this site
(36, 37) ,
presumably by causing the loop to close over the active site, as seen
in the crystal structure of D-ala:D-ala ligase
inhibited by a phospinate substrate analog
(44) . Similarly,
certain combinations of GSP synthetase substrates inhibit its trypsin
cleavage. Maximum protection is observed only when ATP,
Mg
, and GSH are present, under which conditions the
first step of the anticipated catalytic mechanism, formation of the
glutathionylphosphate intermediate, is possible. It is likely that, as
in the other ATP-amide enzymes, protection from proteolysis results
from closing of a flexible loop over the active site to protect the
reactive acylphosphate intermediate from hydrolysis.
The presence of two domains in GSP synthetase/amidase may resolve the puzzle of its high molecular mass, but it raises a second question: why has the protein evolved with opposing catalytic activities? Precedent exists for a bifunctional enzyme with opposing activities. In the case of the glycolytic enzyme, 6-phophofructo-2-kinase/fructose-2,6-bisphosphatase, the activities are reciprocally regulated by protein phosphorylation at a serine residue (48). The human ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase (CD38) synthesizes and degrades cyclic ADP-ribose (49) , which serves as a signaling molecule in pancreatic islets to induce the cells to secrete insulin in response to glucose (50) . In this case, the hydrolase activity is inhibited by ATP (a product of glycolysis) providing a mode for differential regulation (49) . In addition, the opposing activities in this case may provide a mechanism to terminate the signal.
These two precedents suggest that a key feature of GSP synthetase/amidase may be differential regulation of its activities. One factor which might effect regulation is the intracellular pH: the pH optimum of the synthetase activity is 6.8, while our preliminary data suggest that the pH optimum of the amidase activity is higher (see Fig. 5). Indeed, Tabor and Tabor (51) reported that the accumulation of glutathionylspermidine in stationary cultures of E. coli B is favored by a lower medium pH.
With regard to the function of glutathionylspermidine metabolism
in E. coli, the discovery of linked synthesizing and degrading
activities emphasizes possible regulatory roles (e.g. modulation of levels of free spermidine and/or GSH in response to
growth conditions, sparing of GSH and/or spermidine from degradation).
It is clear that glutathionylspermidine metabolism is not essential, as
mutants lacking either GSH or spermidine grow on minimal media
(52-55). Nevertheless, the metabolism may be beneficial under
conditions of environmental stress. To test this possibility, the
phenotypes of gsp mutants exposed to
different forms of stress (oxidant, osmotic, and nutrient) will need to
be examined. Identification of conditions under which
glutathionylspermidine metabolism confers a selective advantage would
provide a clue as to the function of glutathionylspermidine and might
allow the trypanoso-matid homologs of E. coli GSP synthetase
to be cloned by complementation.
Whatever the function of this
metabolism, the presence of both activities in GSP synthetase/amidase
implies that the growth phase-dependent redistribution among levels of
glutathionylspermidine, spermidine, and GSH observed by Tabor and Tabor
(51) may be effected by a single protein. Redistribution among
trypanothione, glutathionylspermidine, GSH, and spermidine has also
been observed in C. fasciculata grown in culture and has been
proposed as a mechanism of growth regulation in trypanosomatids (56).
Given these observations and the high molecular masses of C.
fasciculata GSP and T(SH) synthetases (90 and 82 kDa),
it is tempting to speculate that the trypanosomatid enzymes may also
have both amidase and synthetase activities and that they may effect
the observed growth phase-dependent redistribution among the various
glutathione-spermidine conjugates.
Table:
Summary of purification of GSP
synthetase/amidase from BL21-DE3/pGSP
The reason for the apparent loss and regain of activity in the streptomycin sulfate step is not known.
Table:
Steady-state kinetic constants for spermidine
analogs
In 1-5, the portion of the molecule that is modified from spermidine is boxed. The designations + and - indicate whether the compound detectably inhibits at a concentration of 5 mM, and nd means not determined.
Table:
Steady-state kinetic parameters and
pK values (35) for difluorospermidines
The
values in parentheses were determined in Bis-Tris propane buffer, which
inhibits with respect to spermidine, thereby increasing the apparent
K of (6) into the range where it can be
measured accurately with the coupled
assay.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U23148.