(Received for publication, August 12, 1996, and in revised form, October 9, 1996)
From the ¶ Department of Biochemistry and Molecular Biology,
The Pennsylvania State University, University Park, Pennsylvania 16802, the Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 and
the § Interdepartmental Program in Medicinal Chemistry,
College of Pharmacy and Department of Chemistry, University of
Michigan, Ann Arbor, Michigan 48109
The bifunctional glutathionylspermidine
synthetase/amidase from Escherichia coli catalyzes
both the ATP-dependent formation of an amide
bond between N1 of spermidine
(N-(3-amino)propyl-1,4-diaminobutane) and the glycine carboxylate of glutathione (-Glu-Cys-Gly) and the
opposing hydrolysis of this amide bond (Bollinger, J. M., Jr., Kwon, D. S., Huisman, G. W., Kolter, R., and Walsh, C. T. (1995) J. Biol. Chem. 270, 14031-14041). In our previous work describing
its initial characterization, we proposed that the 619-amino acid (70 kDa) protein might possess separate amidase (N-terminal) and synthetase
(C-terminal) domains. In the present study, we have confirmed this
hypothesis by expression of independently folding and functional
amidase and synthetase modules. A fragment containing the C-terminal
431 amino acids (50 kDa) has synthetase activity only, with
steady-state kinetic parameters similar to the full-length protein. A
fragment containing the N-terminal 225 amino acids (25 kDa) has amidase
activity only and is significantly activated relative to
the full-length protein for hydrolysis of glutathionylspermidine
analogs. This observation suggests that the amidase activity in the
full-length protein is negatively autoregulated. The amidase active
site catalyzes hydrolysis of amide and ester derivatives of glutathione
(e.g. glutathione ethyl ester and glutathione amide) but
lacks activity toward acetylspermidine
(N1 and
N8) and acetylspermine
(N1), indicating that glutathione
provides the primary recognition determinants for
glutathionylspermidine amide bond cleavage. No metal ion is required
for the amidase activity. A tetrahedral phosphonate analogue of
glutathionylspermidine, designed as a mimic of the proposed tetrahedral
intermediate for either reaction, inhibits the synthetase activity
(Ki ~ 10 µM) but does not inhibit
the amidase activity.
The polyamine, spermidine
(N-(3-amino)propyl-1,4-diaminobutane), and the tripeptide,
glutathione (-Glu-Cys-Gly, abbreviated GSH), are present at high
concentrations (0.1-10 mM) in most cells (see, for
example, Refs. 1 and 2 for reviews). With its redox active cysteine
thiol, GSH is the primary small-molecule antioxidant in many cells,
serving to maintain redox poise and reductively scavenge reactive
oxygen species. It is catalytic in these roles by virtue of glutathione
reductase, which maintains GSH in active, reduced form. As a
polycation, spermidine complexes with nucleic acids, proteins, and
phospholipids, thereby influencing their structures and biological
properties (3).
An intriguing link between GSH and spermidine metabolism is found in the protozoal parasites of genera Trypanosoma and Leishmania, including those that cause African sleeping sickness, South American Chagas' disease, and various afflictions known collectively as leishmaniases. In these parasites, it appears that the bis(glutathionyl)spermidine conjugate, trypanothione (see Scheme 1 for structure), has appropriated the important antioxidant roles normally played by GSH (4-10). Thus, these parasites lack GSH reductase and GSH peroxidase activities, but have analogous enzymes that use trypanothione (5, 8-10). Because trypanothione appears to have important roles in these pathogens and is not present in their hosts, its metabolism is an obvious target for design of new antiparasitic drugs.
[View Larger Version of this Image (23K GIF file)]Scheme 1.
The synthesis of trypanothione from glutathione and spermidine is catalyzed by glutathionylspermidine (GSP)1 synthetase and trypanothione synthetase (11, 12). Each couples hydrolysis of ATP (to ADP and Pi) with formation of an amide bond (Scheme 1). The intermediate in this pathway, glutathionylspermidine, was first identified in Escherichia coli more than 3 decades before the discovery of trypanothione itself, and a GSP synthetase activity was partially purified (13, 14). In spite of its early discovery, the physiological role in E. coli of the glutathione-spermidine conjugate is not yet known.
As part of an ongoing investigation of the enzymology and physiology associated with these glutathione-spermidine conjugates, we recently characterized GSP synthetase from E. coli (15). We purified the enzyme, isolated and sequenced its gene, overproduced it, and characterized the recombinant protein. We were surprised to discover that the 70-kDa protein possesses a second catalytic activity: hydrolysis of glutathionylspermidine back to glutathione and spermidine. As the net of these two activities is hydrolysis of ATP (i.e. futile cycling), we proposed that reciprocal regulation of its activities might be an important feature of the bifunctional enzyme. In addition, because the synthetase activity was selectively abrogated by proteolytic cleavage after Arg-538 of the 619-amino acid protein, we proposed that it might possess separate domains for its two activities (N-terminal amidase and C-terminal synthetase) (15).
In the present study, we have confirmed the hypothesis of separate domains by genetic dissection of the bifunctional protein into independently folding and functional amidase and synthetase fragments, and have characterized the fragments with respect to their steady-state kinetic constants. We have evaluated, as an inhibitor of the synthetase activity, a phosphonate analog of glutathionylspermidine designed to mimic the proposed tetrahedral intermediate. Finally, we have begun to address the question of substrate specificity and mechanism of the amidase reaction by 1) evaluating analogs of GSH, spermidine, and glutathionylspermidine as possible substrates and/or inhibitors of the reaction and 2) testing for the presence of a catalytic metal ion.
Preparation of GSP Synthetase/Amidase and Fragments
MaterialsOligonucleotides were purchased from the Harvard Medical School Biological Chemistry and Molecular Pharmacology departmental biopolymer facility or from Integrated DNA Technologies (Coralville, IA). Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA). The vector pET22b and E. coli strain BL21(DE3) were purchased from Novagen (Madison, WI). Construction of the vectors pJMB1 and pGSP has been described previously (15).
Construction of pAMID Vector to Overexpress 25-kDa GSP Amidase FragmentWe had previously (15) subjected the plasmid pJMB1,
which contains a 5.8-kilobase pair insert spanning gsp in
the vector pBluescript (Stratagene), to the transposon mutagenesis
procedure of Berg et al. (16). A mutant plasmid from the
resulting set, with the transposon inserted after nucleotide 675, was
used as template to amplify a 5 fragment of gsp (containing
the amidase domain) using the polymerase chain reaction (PCR). The
primers were 5
-AAGGTAAACATATGAGCAAAGGAACGACCAG-3
(15), which primes for synthesis of the sense strand beginning just 5
of gsp
and the "Res" sequencing primer of Berg et al. (16),
which primes in the transposon for synthesis of the antisense strand of
gsp. The resulting 751-base pair PCR fragment was digested
with NdeI (which cuts in primer 1) and BamHI
(which cuts in the transposon just 3
of gsp) and ligated
into vector pET22b to give pAMID, which encodes an N-terminal fragment
of GSP synthetase/amidase consisting of amino acids 1-225 fused to the
transposon-encoded dipeptide GV. Sequencing of the plasmid by the Dana
Farber Cancer Institute Core Facility confirmed that no mutations were
introduced during PCR amplification.
The vector pSYN was constructed to overproduce a
C-terminal fragment of GSP synthetase/amidase comprising amino acids
189-619. PCR was used to amplify a 1362-base pair, 3 fragment of the
gsp gene. The template for amplification was the vector pGSP
(15), which contains gsp inserted in the plasmid pET22-b
(Novagen) via NdeI (5
) and EcoRI (3
)
restriction sites. Primer 1 (5
-ACCATTCTGGGCCATATGATCCAGACGGAAGAT-3
) corresponds to nucleotides 550-582 of gsp but introduces an
NdeI site (CATATG) for cloning. Primer 2 (5
-ATATTGAATTCTTTGATTAATCCCCGTACTGATTATTC-3
) primes
immediately 3
of gsp for synthesis of the antisense strand. The amplified fragment was digested with NdeI, which cuts
both in primer 1 and at an internal site in gsp after
nucleotide 932. The resulting 367-base pair fragment (nucleotides
565-932) was ligated with NdeI-digested pGSP (two sites
resulting in excision of nucleotides 1-931 of gsp).
Transformants of E. coli strain DH5
containing pSYN (with
the NdeI insert in the desired orientation) were identified
by restriction analysis of plasmid DNA. Sequencing of the plasmid by
the Dana Farber Cancer Institute Core Facility confirmed that no
mutations were introduced during PCR amplification.
E. coli strain
BL21(DE3) transformed with pGSP, pAMID, or pSYN was grown aerobically
in LB medium containing 150 µg/ml ampicillin at 37 °C to an
A600 of 0.6-0.8, when protein expression was
induced by addition of
isopropyl-1-thio--D-galactopyranoside to a final concentration of 500 µM for full-length protein and
amidase fragment or 100 µM for synthetase fragment.
Cultures were incubated an additional 2-4 h and were harvested by
centrifugation. A typical yield was 2.5 g of wet cell paste/liter
of culture.
Full-length GSP synthetase/amidase was purified from strain BL21DE3/pGSP as described previously (15).
In a typical purification of the amidase fragment, cells from 2 liters
of culture were resuspended and lysed as described previously (15).
Treatment with streptomycin sulfate was as described (15). Ammonium
sulfate fractionation steps were as described (15), but the
concentrations were 30% of saturation for the first cut and 70% for
the second cut. The pellet from the 70% ammonium sulfate step was
redissolved in 50 mM Tris-HCl, pH 7.5, 5 mM
DTT, 1 mM EDTA (buffer A) and dialyzed against buffer A. The desalted solution was loaded on a DEAE-Sepharose (Pharmacia Biotech
Inc.) column (2.5 × 25 cm) equilibrated in buffer A. The column
was washed with 100 ml of buffer A, and then developed with a gradient
of NaCl in buffer A (50 ml of 0-100 mM, then 450 ml of
100-400 mM). GSP amidase activity of column fractions was determined by a previously described (15) qualitative assay (thin-layer
electrophoresis with detection by ninhydrin staining). Fractions with
greatest specific activity were combined (78 ml eluting at 230-280
mM NaCl). A 10-ml aliquot of this pool was made 1 M in (NH4)2SO4 by
addition of the solid. This solution was chromatographed in two 5-ml
portions on a Phenyl-Superose HR 10/10 column (Pharmacia) equilibrated
in 20 mM potassium phosphate (pH 7.25), 5 mM
DTT, 1 mM EDTA (buffer B) containing 1 M
(NH4)2SO4. After loading, the
column was washed with 5 ml of buffer A containing 1 M
(NH4)2SO4 and then developed with a
gradient of decreasing (NH4)2SO4
concentration (10 ml of 1.0-0.75 M, then 80 ml of
0.75-0.25 M) in buffer B. Fractions comprising the major
protein peak were combined (20 ml from two injections, eluting at
550-500 mM (NH4)2SO4). The pool was frozen in liquid N2 and stored at 80 °C.
SDS-PAGE analysis of the combined fractions showed the fragment to
be > 90% pure (Fig. 1, lane 4).
For purification of the synthetase fragment, lysis of cells (17 g of
wet cell paste from 6 liters of culture), streptomycin sulfate and
ammonium sulfate fractionation steps, and desalting of redissolved
ammonium sulfate pellet were carried out as described for the
full-length protein (15). The desalted protein was then loaded on a
2.5 × 25-cm DEAE-Sepharose (Pharmacia) column equilibrated in 50 mM Bis-Tris propane·HCl, pH 7.15, 5 mM DTT, 1 mM EDTA (buffer C). The column was developed with a
gradient of NaCl in buffer C (70 ml of 0-100 mM, 610 ml of
100-300 mM), and fractions containing synthetase activity
were pooled (81 ml eluting at 120-180 mM NaCl). This
solution was made 1.2 M in
(NH4)2SO4 by addition of the solid and was chromatographed in 10-ml aliquots on a Phenyl-Superose HR 10/10
column (Pharmacia) equilibrated in 20 mM potassium
phosphate, pH 6.8, 5 mM DTT, 1 mM EDTA (buffer
D) containing 1 M
(NH4)2SO4. The column was developed
with a gradient of decreasing
(NH4)2SO4 concentration (1-0
M) in buffer D. A fraction of the synthetase activity
eluted at the end of this gradient (0 M
(NH4)2SO4), and the remainder
eluted with H2O (free of buffer). The fractions were
combined, frozen in liquid N2, and stored at 80 °C.
SDS-PAGE analysis of the combined fractions showed the fragment to be
>90% pure (Fig. 1, lane 3).
Synthesis of Substrates and Inhibitor
General Synthetic MethodsThese methods were as described previously (17). 1H NMR spectra were recorded at 300 and 360 MHz and are reported in the following manner: chemical shift in ppm downfield from internal tetramethyl silane (multiplicity, integrated intensity, coupling constant in hertz).
MaterialsCommercially available amino acid and dipeptide
precursors were purchased from Bachem or Fisher Scientific.
Boc-alanylglycyl benzyl ester was prepared by a literature procedure
(18); melting point (mp) 86-87 °C (literature mp 85-86 °C; Ref.
18). Trifluoroacetic acid-mediated cleavage of the Boc group afforded
Ala-Gly-OBn (4), which was isolated as its
p-toluenesulfonic acid salt; mp 174.5-175 °C. 1H NMR (D2O) 1.48 (d, 3H, J = 7.25),
2.38 (s, 3H), 4.09 (m, 3H), 5.22 (s, 2H), 7.36 (d, 2H, J = 7.54),
7.44 (s, 5H), 7.72 (d, 2H, J = 8.2). Glutathione ethyl ester was
purchased from Bachem Bioscience Inc. (Switzerland). Other reagents
were obtained from Aldrich unless indicated otherwise.
For the DCC method, a solution of
Z-Glu-OBn (3, 1.0 eq), DCC (Fluka, 1.1 eq) and
hydroxybenzotriazole·H2O (1.2 eq) in DMF (5 ml/mmol
3) was stirred at 0 °C for 10 min. A mixture of the
C-terminal protected dipeptide (·HCl or ·TsOH) (1.0 eq)
and NMM (1.0 eq) in DMF (5 ml/mmol 3) was then added to the
above solution and the reaction mixture was stirred at 0 °C for
1 h, then at room temperature overnight. The precipitated
dicyclohexylurea was removed by filtration and the filtrate was
concentrated in vacuo. For the MCCA method, to a stirred
solution of Z-Glu-OBn (3, 1.0 eq) in dry tetrahydrofuran (10 ml/mmol 3) was added NMM (1.0 eq), followed by
i-butylchloroformate (1.1 eq) at 25 °C under dry
nitrogen. Stirring was continued at
25 °C for 15 min, then a
mixture of NMM (1.1 eq) and the C-terminal protected dipeptide
(·HCl or ·TsOH) (1.0 eq) in dry tetrahydrofuran (20 ml/mmol 3) was added. The reaction mixture was allowed to
stir at
20 °C for 1 h and at room temperature for 24 h.
Volatile components were removed under reduced pressure.
Scheme 2.
Z-
6
was synthesized from 3 and 4 by both the DCC and
MCCA method (yields 93% and 82%, respectively). After concentration
of the reaction mixture, the residue was suspended in EtOAc and washed
successively with 5% citric acid, H2O, 5% NaHCO3, and brine. Removal of the solvent afforded a solid
product, which was crystallized from EtOAc/hexane to give a white
powder, mp 142-144 °C (Literature 140-142 °C; Ref. 19).
RF 0.55 (4:1 CHCl3/MeOH); 1H
NMR (CDCl3) 1.40 (d, 3H, J = 6.71), 1.80 (dm, 2H),
2.15-2,30 (m, 2H), 3.90-4.10 (m, 2H), 4.32-4.51 (m, 2H), 5.10 (s,
2H), 5.15 (s, 2H), 5.20 (s, 2H), 5.70 (d, 1H, J = 6.82), 6.22 (d,
1H, J = 5), 6.78 (br, 1H), 7.40 (s, 15H); 13C NMR
(CDCl3)
172.5, 172.2, 172.0, 169.5, 164.8, 128.8, 128.7, 128.5, 128.3, 128.2, 67.6, 67.4, 67.3, 54.0, 49.0, 41.6, 32.3, 29.2, and 17.9.
A suspension of 6 (0.48 g, 0.81 mmol) and 10% Pd/C (100 mg) in a mixture of EtOH (80 ml)
and MeOH (25 ml) was shaken on a Parr hydrogenator at 40 p.s.i.
for 16 h. The catalyst was removed by filtration. The filtrate was
concentrated, and the resultant hygroscopic solid material was purified
by ion-exchange chromatography on DEAE-cellulose using 0.1 M NH4HCO3 (pH 7.8) as eluant. A
white crystalline product (hygroscopic) was obtained in quantitative
yield; mp 195-197 °C (literature mp: 193-195 °C; Ref. 18);
1H NMR (methanol-d4) 1.35 (d,
3H, J = 7.2), 2.05-2.15 (m, 2H), 2.45-2.55 (m, 2H), 3.62 (t,
1H), 3.71-3.84 (m, 2H), 4.30-4.40 (m, 1H); 13C NMR
(methanol-d4)
175.7, 174.4, 172.9, 171.6, 53.8, 50.7, 41.9, 32.6, 27.1, and 18.1.
7 was synthesized from Z-Glu-OBn and
Ala-Gly-NH2·HCl by both the MCCA and DCC methods with
yields of 22% and 78%, respectively. The resultant syrupy residue was
triturated with EtOAc. A yellow solid was obtained, which was
triturated successively with 5% citric acid, H2O, 5%
NaHCO3, and acetone:Et2O (1:1 v/v). A white powder was obtained; a portion of this product was recrystallized from
MeOH, mp 193-194 °C; RF 0.71 (4:1:5
BuOH:H2O:AcOH, upper phase); 1H NMR
(CD3OD) 1.17 (d, 3H, J = 7.1), 1.70-2.05 (dm,
2H), 2.25 (t, 2H), 3.42-3.68 (m, 2H), 4.05-4.25 (m, 2H), 5.0-5.1 (m,
2H), 5.12 (s, 2H), 7.06 (s, 1H), 7.16 (s, 1H), 7.21-7.49 (m, 10H), 7.79 (d, 1H, J = 7, 6), 8.05-8.15 (m, 2H); 13C
NMR(CD3OD)
176.10, 175.1, 174.6, 174.0, 154.2, 129.7, 129.3, 129.0, 68.1, 67.9, 55.2, 51.2, 43.4, 32.8, 28.2, and 17.6.
2 was prepared by two methods. In
method 1, a mixture of 7 (0.1 g, 0.2 mmol) and 10% Pd/C (50 mg) in MeOH (25 ml) was shaken on a Parr hydrogenator (40 p.s.i.) for
20 h. The catalyst was removed by filtration and the filtrate was
concentrated to afford a syrupy material, which was triturated with
acetone and a minimum amount of MeOH. The resultant white solid product was recrystallized from MeOH-Et2O to afford a white
crystalline material (0.05 g, 87%); mp 194-195.5 °C;
RF 0.33 (MeOH:EtOAc:AcOH = 5:4:1);
1H NMR (CD3OD) 1.41 (d, 3H, J = 6.8),
2.10-2.25 (m, 2H), 2.52-2.61 (m, 2H), 3.62-3.73 (m, 1H), 3.87 (s,
2H), 4.25-4.35 (q, 1H); 13C NMR (CD3OD)
176.2, 175.8, 175.1, 174.0, 55.4, 51.4, 43.4, 32.7, 27.6, 17.5; mass
spectroscopy (fast atom bombardment) m/z 275 (MH+, 100), 177 (20.3), 155 (23), 119 (18), 85 (38); high
resolution mass spectrum (fast atom bombardment) calculated for
C10H18N4O5H (MH+) 275.1355, found 275.1351. For method 2, a solution of
H-Ala-Gly-NH2 (1.01 g, 5.6 mmol), Boc-Glu(OSu)-OtBu (2.24 g, 5.6 mmol), and N-methylmorpholine (1.3 ml) in DMF (80 ml)
was stirred for 10 h at room temperature. The solvent was
evaporated in vacuo, and the residue was partitioned between
0.1 N HCl and EtOAc. The organic layer was washed with
saturated NaHCO3(aq) and brine, dried over MgSO4, and evaporated to dryness. The residue was
chromatographed on silica gel with CHCl3-MeOH (12:1) to
give Boc-
-Glu(
-OtBu)-Ala-Gly-NH2 (2.04 g) in 85%
yield. Treatment of the protected tripeptide with 50% trifluoroacetic
acid in CH2Cl2 under N2 for 30 min,
evaporation of the solvent, and chromatography on Sephadex G-10
afforded
-Glu-Ala-Gly-NH2 in 88% yield. TLC analysis of
the product (n-PrOH/AcOH/H2O:10/1/5) on
silica gave one spot which comigrated with the material synthesized and
characterized by Method 1 above, and the 1H NMR spectra of
the products from the two methods were identical.
GSH amide was prepared by ammonolysis of glutathione ethyl ester. In a typical reaction, 10 ml of 2 M NH3 in methanol (Aldrich) was cannulated under N2 into a sealed, N2-purged vessel containing 0.10 g of glutathione ethyl ester, 0.052 g of dithiothreitol, and a single crystal of dimethylaminopyridine (Sigma) as catalyst. The solution was incubated at 42 °C for 72 h. The reaction was monitored by thin-layer chromatography on silica with CH3Cl:MeOH:33% acetic acid (5:3:1) in water mobile phase (RF values: glutathione ethyl ester, 0.6; glutathione methyl ester, 0.5; product, 0.2). After 72 h, a 0.2-ml aliquot of 1 M dithiothreitol in methanol was added to the reaction, and the solvent was evaporated in vacuo. The solid was redissolved in 5 ml of 0.1% trifluoroacetic acid in H2O. The solution was filtered through a column of 1.5 ml of Dowex AG-50 (Na+ form, Bio-Rad) to remove remaining NH4+. Fractions containing product (flow-through) were then filtered through a C18 spice cartridge (Rainin) to remove oxidized and reduced dithiothreitol (product elutes in the void volume, while dithiothreitol is retarded). Fractions containing product were evaporated to dryness in vacuo, and product was redissolved in 10 mM dithiothreitol in H2O for use. The product with RF = 0.2 was quantitatively converted to a species that comigrated with GSH by treatment with GSP synthetase/amidase. In this conversion, ammonia was produced, as detected by the glutamate dehydrogenase spectrophotometric assay described below. The quantity of NH3 released following exhaustive hydrolysis was assumed to be equal to the quantity of glutathione amide originally present.
Phosphonate Analog (8) of GlutathionylspermidineThe hydroxyspermidine-containing phosphopeptide, 8 (see "Results" for structure), was prepared in a convergent synthesis based on the retrosynthetic pathway outlined previously (17). The detailed syntheses of 8 and several related phosphopeptides, together with more extensive inhibition studies, will be published elsewhere.2
Characterization of GSP Synthetase/Amidase and Fragments
Materials35S-Glutathionylspermidine was prepared enzymatically from [35S]glutathione (New England Nuclear) as described previously (15). N1-glutathionylspermidine disulfide was purchased from Bachem Bioscience Inc. (Switzerland). ATP, glutathione, spermidine, NADH, NAD, NADPH, phospho(enol)pyruvate, 2-oxoglutarate, dithiothreitol, glutamate dehydrogenase from Proteus species (product no. G-4387), and alcohol dehydrogenase from Bakers' Yeast (product no. A-3263) were purchased from Sigma. Aldehyde dehydrogenase from yeast (product no. 171832), and pyruvate kinase and lactate dehydrogenase (the latter two in an equi-unit suspension in 3.2 M ammonium sulfate) were purchased from Boehringer Mannheim.
Protein AssaysProtein concentrations were determined by the method of Bradford as supplied by Bio-Rad. Bovine serum albumin (Pierce) was used as standard.
Enzyme Activity AssaysGSP synthetase activity was measured by a previously described coupled assay (15). Amidase activity toward glutathionylspermidine was assayed radiometrically by monitoring conversion of [35S]GSP to [35S]GSH by a previously described thin-layer electrophoresis assay (15).
GSP amidase-catalyzed hydrolysis of glutathione ethyl ester was assayed
by coupling production of ethanol to reduction of NAD through the
activities of alcohol dehydrogenase and aldehyde dehydrogenase. In a
final volume of 400 µl, the assay contained 50 mM
Tris-HCl, pH 8.2, 1 mM NAD, 500 µM DTT, 0.5 mg (170 activity units) of alcohol dehydrogenase, 0.07 mg (4 activity
units) of aldehyde dehydrogenase, either 6.6 ng of GSP amidase fragment or 66 ng of full-length GSP synthetase/amidase, and varying amounts of
GSH ethyl ester. GSH ethyl ester has a significant background rate of
hydrolysis at pH 8.2 (kobs ~ 3 × 104 min
1), so this rate was measured at
each concentration prior to initiation of the reaction with the amidase
fragment or full-length protein. The (increasing) absorbance at 340 nm
was monitored. By subjecting limiting quantities of GSH ethyl ester to
complete hydrolysis, the assay stoichiometry was verified to be 2 mol
of NADH produced (in oxidation of ethanol to acetate)/mol of GSH ethyl
ester hydrolyzed.
GSP amidase-catalyzed hydrolysis of glutathione amide and
-Glu-Ala-Gly-NH2 was assayed by coupling production of
NH3 to oxidation of NADPH through the activity of glutamate
dehydrogenase. In a final volume of 400 µl, the assay contained 50 mM Tris-HCl, pH 8.2, 1 mM 2-oxoglutarate, 200 µM NADPH, 64 µg of glutamate dehydrogenase, either 50 ng of GSP amidase fragment or 800 ng of full-length protein, and
varying amounts of GSH amide. The reaction was initiated by addition of
substrate, and the (decreasing) absorbance at 340 nm was monitored.
Solutions containing 320 µM GSP synthetase/amidase or GSP amidase fragment were dialyzed exhaustively against 50 mM HEPES (pH 7.5), 5 mM DTT, 1 mM EDTA. Catalytic activity was verified after dialysis. A 0.5-ml aliquot of each protein sample and of the dialysis buffer was mixed with 0.5 ml of 4 N HCl (J.T. Baker Ultrex Ultrapure Reagent), and the samples were incubated in sealed tubes at > 90 °C for 12 min with periodic vigorous mixing. Insoluble protein residue was removed by centrifugation, and the supernatant of each sample was submitted to the Chemical Analysis Laboratory of the University of Georgia (Athens, GA) for inductively coupled plasma emission spectroscopy.
Dissection of Bifunctional Protein into Separate Domains
Preparation of Amidase Fragment and Comparison to Full-length ProteinOur previous finding that trypsin cleavage of GSP synthetase/amidase after Arg-538 gives a 61.6-kDa N-terminal fragment with only amidase activity suggested that the protein might possess independent amidase and synthetase domains (15), and led us to test whether a smaller N-terminal fragment might independently fold into a functional amidase domain. We used a previously constructed set of transposon insertional mutants of gsp in the high copy number plasmid pBluescript (15). Several plasmids with the transposon disrupting gsp still gave rise to ~ 50-fold greater amidase activity in crude lysates than a pUC19 control plasmid. Of these, one with the transposon inserted after nucleotide 675 encoded the smallest N-terminal fragment, amino acids 1-225 of GSP synthetase/amidase fused to the transposon-encoded dipeptide GV. A plasmid with the transposon inserted after nucleotide 207 did not give rise to increased activity, suggesting that the minimal N-terminal fragment needed for amidase activity is between 69 and 225 amino acids. The 225-amino acid N-terminal fragment was overproduced, and was purified to ~ 95% homogeneity (Fig. 1, lane 4) by the same two-column procedure employed for the full-length protein. The fragment showed no evidence of insolubility or instability during this procedure. The yield corresponded to 66 mg/liter culture.
Crude estimates of the steady-state kinetic parameters
(kcat and Km) for hydrolysis
of glutathionylspermidine by the amidase fragment were obtained by the
thin-layer electrophoresis assay, and these data suggested that
liberation from the C-terminal 394 amino acids significantly
activates the amidase domain (severalfold greater
kcat/Km than for the
full-length protein). Kinetic parameters were subsequently measured for
several analogues of glutathionylspermidine (-Glu-Cys-Gly-OEt,
-Glu-Cys-Gly-NH2,
-Glu-Ala-Gly-NH2,
-Glu-Ala-Gly-p-nitroanilide) for which colorimetric assays allowed more precise determinations to be made (see section below on substrate specificity studies).
The recent discovery that the tertiary folds of
D-alanine:D-alanine ligase and glutathione
synthetase from E. coli are closely related despite minimal
similarity in their primary structures (20) suggested that this fold
might be characteristic of a family of bacterial ATP-cleaving
(ADP-forming), amide bond-forming enzymes. D-ala:D-Ala ligase and GSH synthetase are
composed of ~ 310 amino acids each, and we considered this as an
estimate for the extent of the synthetase domain of GSP
synthetase/amidase. A second consideration in designing a
synthetase-only construct was the sequence similarity among the
C-terminal ~ 370 amino acids of GSP synthetase/amidase and the
hypothetical protein products of three other bacterial open reading
frames, ygiC and yjfC from E. coli and
ygiC from Haemophilus influenzae (Fig.
2). These hypothetical proteins may be related
ATP-cleaving, amide bond-forming enzymes, and the region of homology
may therefore delimit the synthetase domain. On the basis of these
considerations, two potential synthetase constructs were prepared. A
fragment containing the C-terminal 318 amino acids (beginning with
Met-312), which does not span the entire homology region, was found to
be insoluble upon overexpression and to lack detectable synthetase
activity. In contrast, a fragment of 431 amino acids (beginning at
Met-189) is soluble and active for ATP-dependent
glutathionylspermidine synthesis. This fragment was purified to ~ 90% homogeneity (Fig. 1, lane 3) by the same two-column
procedure employed for the recombinant, full-length protein. The
protein eluted from the hydrophobic interaction matrix, Phenyl-Superose, in two fractions, the first eluting with column buffer
containing no (NH4)2SO4 and the
second eluting with H2O (requiring removal of even the
buffer salt). This strong interaction suggests that the synthetase
fragment has surface-exposed hydrophobic residues not displayed in the
full-length protein.
Steady-state kinetic parameters for the synthetase fragment were determined (Table I). The fragment has only slightly (~3-fold) reduced kcat relative to the full-length protein and similar specificity constants for its three substrates. Thus, the C-terminal 50-kDa fragment folds autonomously into a fully functional amide-forming domain.
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Substrate Specificity Studies
Specificity of Synthetase ActivityA main objective of our
ongoing investigation of GSP synthetases from E. coli and
Trypanosomatidae has been to design GSP analogs as
inhibitors that might be useful as mechanistic and physiological
probes. In design of such analogs, we hoped to substitute a redox inert
residue for Cys in the GSH portion of the molecule, in order to avoid
synthetic problems involving thiol redox chemistry. To assess whether
such a substitution would diminish substrate/inhibitor specificity, we
examined -Glu-Ala-Gly as a substrate of the synthetase (Table I).
The GSH analog is a reasonable substrate, with
kcat (6.4 s
1) identical with that
of GSH and Km (10 mM) elevated by ~ 14-fold.
In order to define
recognition determinants for the amidase activity, potentially
hydrolyzable derivatives of glutathione and spermidine were tested as
substrates of both the full-length protein and the amidase fragment
(Table II). Both the simple amide and the ethyl ester of
GSH are good substrates, while no hydrolysis of
N1-acetylspermidine,
N8-acetylspermidine, or
N1-acetylspermine was detected by the
qualitative electrophoresis assay following a 100-min incubation under
conditions (10 mM substrate, 3.2 µM amidase
fragment, pH 8.2, 37 °C) which gave > 90% hydrolysis of
glutathionylspermidine in 5 min. The cysteine sulfhydryl group is not a
crucial recognition determinant, as its substitution by a proton in
-Glu-Ala-Gly-NH2 results in only 10-fold loss in
kcat/Km for the amidase
fragment and a 2-3-fold increase for the full-length protein. As is
often true of amidases, the ester derivative is more efficiently
cleaved than the two amide substrates. For example,
kcat/Km for hydrolysis of
-Glu-Cys-Gly-OEt by full-length protein is ~ 40-fold greater
than for hydrolysis of glutathionylspermidine (15), arising from a
greater kcat. Somewhat surprisingly, the simple
amide of glutathione is hydrolyzed with a much greater (~ 20-fold) kcat than glutathionylspermidine, though its Km also increases somewhat.
These data indicate that the amidase active site
recognizes predominantly the glutathione portion of
glutathionylspermidine.
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The kinetic parameters for the various GSH analogues in Table II also
demonstrate that the amidase fragment is indeed activated relative to
the full-length protein. Depending on the substrate, kcat/Km for the fragment is
between 3-fold (-Glu-Ala-Gly-NH2) and 74-fold
(
-Glu-Ala-Gly-p-nitroanilide) greater than for the full-length protein. These data suggest that the amidase domain is
negatively autoregulated in the context of the full-length protein.
Inhibitor Design and Testing
Phosphonate Analog of GSP as Inhibitor of Synthetase ActivityThose ATP-cleaving (ADP-forming), amide bond-forming
enzymes that have been well characterized are believed to employ a
mechanism (Scheme 3, A) involving phosphoryl
transfer from ATP to the carboxylate oxygen to form an acyl phosphate
(9), attack on this intermediate by the amine, and
decomposition of the resulting tetrahedral adduct (10) by
elimination of phosphate (21-23). There is extensive precedent for
potent inhibition of several of these, including D-alanine:D-alanine ligase (24-26), glutamine
synthetase (27-29), and glutathione synthetase (30), by phosphonate
(11) and phosphinate (12) analogs of the
corresponding substrates. Some of these analogs can undergo
enzyme-mediated phosphoryl transfer from ATP to the phosphon(phin)ate O
(a step that is typically slow, leading to slow-binding inhibition
kinetics), to produce species akin to 13 that (presumably)
closely mimic the normal tetrahedral intermediates (Scheme 3,
B) and inhibit with high affinity (nanomolar
KD) due to their extremely slow dissociation (24,
27, 28, 31). On the basis of these precedents, we designed the
hydroxyspermidine-containing phosphopeptide, 8, as a
potential inhibitor of GSP synthetase, with the aforementioned Ala Cys substitution incorporated for synthetic convenience (see Scheme
3).
Phosphonate 8 is a potent inhibitor of GSP synthetase
activity with respect to the substrate GSH (Fig. 3).
Analysis of Fig. 3 according to non-competitive or mixed-type
inhibition (Scheme 4) gave inhibition constants of 6 µM for inhibitor binding to free enzyme
(KI) and 14 µM for binding to the
enzyme-GSH complex (KI). No
time-dependent (slow-binding) inhibition was observed (Fig.
4), nor was 8 found to stimulate uncoupled ATP hydrolysis by the synthetase. These results suggest that
phosphorylation of 8 is not occurring, and, therefore, that
the inhibitor may be acting as a simple bisubstrate analog rather than
as an intermediate or transition state mimic.
Scheme 4.
Phosphonate Analog of GSP as Inhibitor of Amidase Activity
In
addition to their use as slow-binding inhibitors of amide bond-forming
enzymes, phosphapeptides have also been used as amidase inhibitors (32,
33). Thus, for amidases that facilitate direct attack of
H2O (e.g. zinc or aspartic proteases), a
tetrahedral species akin to 14 is an intermediate (Scheme
5), and phosphapeptides analogous to 8 have
been observed to bind tightly as mimics (33, 34). Phosphonate
8 was therefore tested for inhibition of amidase-catalyzed
-Glu-Cys-Gly-NH2 hydrolysis. In the presence of 100 µM substrate, 1.5 mM 8 had no
discernible effect on the amidase activity of either the full-length
protein or the amidase fragment. Thus, 8 is at best a poor
(high mM Ki) inhibitor of GSP
amidase.
Scheme 5.
Analysis for Metal Ions in GSP Synthetase/Amidase and the Amidase Fragment
In order to test for the presence of a catalytic metal ion in the amidase active site, the metal ion contents of GSP synthetase/amidase and amidase fragment were determined. Neither contains significant quantities of zinc, iron, manganese, cobalt, or nickel, though stoichiometric Ca2+ was found in both. As Ca2+ is not known to act as a cofactor for amide hydrolysis, these observations suggest that a metal ion is not required for GSP amidase catalysis.
The above results demonstrate that the two activities of glutathionylspermidine synthetase/amidase reside in independently folding and functional domains, suggesting that the protein evolved by fusion of amidase and synthetase fragments. By their fusion, any possibility for differential regulation of the component activities of this potential futile cycle at the level of transcription or translation is seemingly eliminated (although production of alternative mRNAs is formally possible). Assuming that the physiological function of the protein does not derive from futile ATP consumption, the two activities are probably differentially regulated post-translationally, either by an allosteric mechanism or by covalent modification. The observation by Tabor and Tabor (35) that glutathionylspermidine accumulates in saturated, anaerobic cultures of E. coli B grown in glucose-rich medium and is rapidly (in less than 5 min) and completely hydrolyzed following dilution into fresh medium lends credence to the suggestion of physiological regulation. The increased amidase activity of the N-terminal fragment relative to the full-length protein suggests one possible mechanism for differential regulation; if this activity is inhibited in the context of the full-length protein, relief of inhibition either by proteolysis or by a conformational change upon ligand binding could selectively enhance amidase activity.
The selective advantage (if any) conferred to E. coli by this unique bifunctional enzyme remains an enigma. We previously proposed that the enzyme might serve to modulate levels of free spermidine or glutathione (15). If so, the presence of synthetase and amidase activities would allow for a bidirectional response (increase or decrease in concentration) without requirement for new protein synthesis or for synthesis or degradation of the relevant metabolite. In addition, in regulating levels of free spermidine, the specificity of the amidase for glutathione-containing amides would render this system orthogonal with the spermidine acetyltransferase system (36).
The amino acid sequence of the amidase domain provides no clue as to catalytic mechanism, as it lacks similarity with any known protein sequence. The amidase may therefore function either by acid/base-assisted direct attack of H2O (as do the aspartic and zinc proteases) or by covalent catalysis (as do the serine and cysteine proteases). Two observations suggest that the latter is more likely. First, the domain lacks a catalytic metal ion. Second, the phosphonate 8, which should closely mimic the tetrahedral intermediate for direct attack by H2O, does not inhibit the amidase. We are currently using chromogenic ester derivatives of glutathione and rapid kinetic methods to search for a glutathionyl enzyme intermediate.