Identification of Critical, Conserved Vicinal Aspartate
Residues in Mammalian and Bacterial ADP-ribosylarginine
Hydrolases*
Piotr
Konczalik
and
Joel
Moss
From the Pulmonary-Critical Care Medicine Branch, NHLBI, National
Institutes of Health, Bethesda, Maryland 20892-1434
 |
ABSTRACT |
NAD:arginine ADP-ribosyltransferases and
ADP-ribosylarginine hydrolases catalyze opposing arms of a putative
ADP-ribosylation cycle. ADP-ribosylarginine hydrolases from mammalian
tissues and Rhodospirillum rubrum exhibit three regions of
similarity in deduced amino acid sequence. We postulated that amino
acids in these consensus regions could be critical for hydrolase
function. To test this hypothesis, hydrolase, cloned from rat brain,
was expressed as a glutathione S-transferase fusion protein
in Escherichia coli and purified by glutathione-Sepharose
affinity chromatography. Conserved amino acids in each of these regions
were altered by site-directed mutagenesis. Replacement of Asp-60 or
Asp-61 with Ala, Gln, or Asn, but not Glu, significantly reduced enzyme
activity. The double Asp-60
Glu/Asp-61
Glu mutant was inactive, as were Asp-60
Gln/Asp-61
Gln or Asp-60
Asn/Asp-61
Asn. The catalytically inactive single and double mutants appeared to retain conformation, since they bound ADP-ribose, a substrate analogue and an inhibitor of
enzyme activity, with affinity similar to that of the wild-type hydrolase and with the expected stoichiometry of one. Replacing His-65,
Arg-139, Asp-285, which are also located in the conserved regions, with
alanine did not change specific activity. These data clearly show that
the conserved vicinal aspartates 60 and 61 in rat ADP-ribosylarginine
hydrolase are critical for catalytic activity, but not for high
affinity binding of the substrate analogue, ADP-ribose.
 |
INTRODUCTION |
Mono-ADP-ribosylation is a post-translational modification of
proteins, in which the ADP-ribose moiety of
-NAD is transferred to
specific amino acid residues in target proteins (1). This reaction has
been well characterized for bacterial toxins, which thereby alter the
activity of critical regulatory proteins in mammalian cells (1).
Cholera toxin, for example, ADP-ribosylates an arginine in the
-subunit of the stimulatory guanine nucleotide-binding (G) protein
of the adenylyl cyclase system, resulting in its activation and an
increase in intracellular cAMP (2). Pertussis toxin modifies a cysteine
in another family of G proteins, blocking the action of inhibitory
agonists on adenylyl cyclase (3, 4). Other toxins use different
proteins, and in some instances, different acceptor amino acids
(e.g. asparagine) as substrates for ADP-ribosylation (2-7).
Enzymes that catalyze reactions similar to bacterial toxins have been
identified in mammalian and avian tissues, including turkey
erythrocytes, chicken neutrophils, rat neurons, and mammalian cardiac
and skeletal muscle cells (8-10). These NAD:arginine
ADP-ribosyltransferases vary in cellular localization, some being
intracellular, others secreted, and a third group linked to the cell
surface by glycosylphosphatidylinositol anchors (8). These transferases
catalyze the stereospecific transfer of ADP-ribose from
-NAD to the
guanidino group of arginine (protein), producing the
-ADP-ribosylarginine (protein) (11).
In some instances, substrates and/or effectors of ADP-ribosylation have
been defined. In murine cytotoxic T cells, a
glycosylphosphatidylinositol-anchored transferase regulates
proliferation and cytotoxic activity, possibly by modulating activity
of the protein tyrosine kinase p56lck, through ADP-ribosylation
of a regulatory protein, p40 (12). Integrin
7 is a major
substrate of a glycosylphosphatidylinositol-linked transferase in
skeletal muscle cells (C2C12) (13), which, based on inhibitor studies,
was proposed to play a role in muscle cell differentiation (14).
Integrin
7
1, a laminin-binding protein, may be involved in cell adhesion and communication between myoblasts and extracellular matrix (15-17).
ADP-ribosylation of arginine residues may be a reversible modification
of proteins. ADP-ribosylarginine hydrolases cleave the
ADP-ribose-arginine (protein) linkage, regenerating the arginine guanidino group (18). The hydrolase-catalyzed reaction is
stereospecific and utilizes
-ADP-ribosylarginine, a product of the
transferase-catalyzed reaction (19). Hydrolases thus complete an
ADP-ribosylation cycle that could reversibly regulate the function of
substrate proteins (19).
In the photosynthetic bacterium Rhodospirillum rubrum
(20), an ADP-ribosylation cycle plays an important role in nitrogen fixation, which is controlled by the reversible ADP-ribosylation of
dinitrogenase reductase. An ADP-ribosyltransferase (termed DRAT for
dinitrogenase reductase ADP-ribosyltransferase) inactivates dinitrogenase reductase by ADP-ribosylation, and DRAG or
dinitroreductase ADP-ribosylarginine glycohydrolase regenerates the
active form by releasing ADP-ribose from the enzyme (20). This
ADP-ribosylation cycle is regulated by environmental signals
(e.g. nutrients, light) that determine the requirement for
nitrogen fixation and hence the need for active or inactive
dinitrogenase reductase (21, 22).
In eukaryotes, ADP-ribosylarginine hydrolases have been identified in
mammalian and avian tissues. ADP-ribosylarginine hydrolase cDNAs
cloned from human, rat, and mouse tissues share >82% similarity of
deduced amino acid sequences (23, 24). Consistent with this degree of
similarity, rabbit anti-rat brain hydrolase polyclonal antibodies
cross-react with hydrolases from turkey erythrocytes, and calf, mouse,
and human brains (24). Despite their functional similarities,
ADP-ribosylarginine hydrolases from human, rat, and mouse tissues
and R. rubrum exhibit only limited regions of similarity in
deduced amino acid sequences (24). We postulated, therefore, that amino
acids in these areas could represent consensus motif residues critical
for hydrolase function. We report here that mutation of rat brain
ADP-ribosylarginine by replacement of the conserved Asp-60 and/or
Asp-61 with alanine, glutamine, or asparagine significantly reduced
enzyme activity, but not the affinity for a substrate analogue
ADP-ribose, consistent with an essential role for both residues in
catalysis, but not structure of the substrate site.
 |
EXPERIMENTAL PROCEDURES |
Materials--
[adenine-U-14C]NAD (252 mCi/mmol) was purchased from Amersham Pharmacia Biotech;
[U-14C]L-arginine (320 mCi/mmol) from NEN
Life Science Products;
-NAD from Sigma; Affi-Gel 601 (boronate) and
prestained protein SDS-polyacrylamide gel electrophoresis standards
from Bio-Rad; plasmid DNA isolation QIAprep Spin Miniprep Kit from
Qiagen; QuikChange site-directed mutagenesis kit from
Stratagene; ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction
Kit with AmpliTaq DNA Polymerase FS from Perkin-Elmer;
glutathione-Sepharose 4B from Amersham Pharmacia Biotech; cholera toxin
A subunit from List Biological Laboratories Inc.; and
isopropyl-1-thio-
-D-galactopyranoside from Gold
Biotechnology, Inc. Restriction enzymes were purchased from Roche
Molecular Biochemicals. Other reagents were of analytical grade.
Construction of Wild-type and Mutant Rat Brain
ADP-ribosylarginine Hydrolase Expression Vectors--
Wild-type
hydrolase cDNA was amplified from the
ZAP clone (23) with
forward and reverse primers 5'-ACGTACGTGGATCCATGGGTGGGGGCCTGA-3' and 5'-ACGTACGTGAATTCCCTAGGAACCTAGTATAGTG-3', respectively, digested with BamHI and EcoRI, and cloned into a pGEX-2T
expression vector (Amersham Pharmacia Biotech), to produce the rWT
plasmid. Pfu polymerase was used for polymerase chain
reaction (Perkin-Elmer Thermal Cycler) amplification according to the
manufacturer's protocols for 25 cycles of 96 °C, 45 s;
62 °C, 1 min; 72 °C, 2 min. Mutants were generated by the
QuikChange site-directed mutagenesis method (Stratagene) according to
the manufacturer's protocol. The following oligonucleotides and the
complementary primers (not shown) were used to generate hydrolase
mutants. The modified codons are underlined, and the mutant is
identified in parenthesis:
5'-GGAGAGTCAGCGCCGACACCATCATGC-3' (D60A),
5'-GAGAGTCAGCGTGCCACCATCATGCAC-3' (D61A),
5'-GATGACACCATCATGGCCCTGGCTACAGC-3' (H65A),
5'-GGAGCCGCCATGGCCGCCATGTGCATCG-3' (R139A),
5'-CCATGATTGCCTATGGCGCCCTCCTGGC-3' (D285A),
5'-GGAGAGTCAGCGAGGACACCATCATGC-3' (D60E),
5'-GGAGAGTCAGCAATGACACCATCATGC-3' (D60N),
5'-GGAGAGTCAGCCAGGACACCATCATGC-3' (D60Q),
5'-GGAGAGTCAGCGATGAGACCATCATGC-3' (D61E),
5'-GGAGAGTCAGCGATAATACCATCATGC-3' (D61N),
5'-GGAGAGTCAGCGATCAGACCATCATGC-3' (D61Q),
5'-GGAGAGTCAGCGCCGCAACCATCATGCAC-3' (D60A/D61A),
5'-GGAGAGTAGCGAGGAGACCATCATGCAC-3' (D60E/D61E),
5'-GGAGAGTCAGCAACAACACCATCATGCAC-3' (D60N/D61N), 5'-GGAGAGTCAGCCAGCAGACCATCATGCAC-3' (D60Q/D61Q).
After amplification, parental, supercoiled dsDNA was digested with
DpnI endonuclease (2.5 units for 2 h at 37 °C in 50 µl of reaction mix). DpnI endonuclease, which is specific
for methylated and hemimethylated DNA, was used to digest the parental
DNA template and thereby select for DNA containing the desired
mutation. DNA isolated from almost all Escherichia coli
strains is dam-methylated and therefore susceptible to
DpnI digestion (25). The nicked vector DNA containing the
hydrolase mutations was transfected into Epicurian Coli XL1-Blue
supercompetent cells. Transformed Epicurian Coli XL1-Blue
supercompetent cells were plated on LB-ampicillin agar plates that were
incubated at 37 °C overnight. 20 colonies of each mutant were grown
overnight in 3 ml of LB/Amp at 37 °C. Plasmid cDNA was isolated
by QIAprep Spin Miniprep Kit (Qiagen), and the entire coding region was
sequenced (ABI PRISM 377, Perkin-Elmer). Proteins were synthesized in
E. coli BL21(DE3) with incubation at 30 °C for 12 h
in the presence of 0.2 mM
isopropyl-1-thio-
-D-galactopyranoside (100 ml total
volume). Cells were harvested, washed with ice-cold phosphate-buffered
saline, and suspended in 5 ml of lysis buffer (10 mM sodium
phosphate, 10 mM EDTA, pH 8.0). After freezing (dry ice, 5 min) and thawing at room temperature (a total of three times) followed
by sonicating for 1 min on ice, the cell lysate was centrifuged
(4000 × g, 20 min), and the supernatant was incubated with gentle agitation at room temp for 30 min with a slurry of glutathione-Sepharose 4B equilibrated with phosphate-buffered saline.
The matrix was washed before fusion proteins were eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH
8.0, and concentrated in Microcon microconcentrators to a final volume of 500 µl. Samples (20 µl) were mixed with 10 µl of 2×
SDS/sample buffer (4% SDS, 10% glycerol, 0.02% bromphenol blue, 1%
-mercaptoethanol, 125 mM Tris, pH 6.8), boiled for 3 min, and subjected to electrophoresis in 4-20% gradient Tris-glycine
gels (Novex), which were then stained with 0.05% Coomassie Blue.
Fusion proteins were estimated to be ~95% pure. Protein
concentration was quantified using bovine serum albumin as standard.
ADP-ribosylarginine Hydrolase Assay--
The substrate was
synthesized by incubation at 30 °C overnight in 2 mM
-NAD, 2 mM [14C]arginine (50 µCi), 50 µg of cholera toxin A subunit, 50 µg of recombinant human
ADP-ribosylation factor (rARF 1; gift from Walter Patton, NHLBI,
National Institutes of Health, Bethesda, MD), 30 µg of ovalbumin, and
5 µg of cardiolipin, 20 mM dithiothreitol, 100 µM GTP, 50 mM potassium phosphate, and 10 mM MgCl2, pH 7.5 (total volume 300 µl).
ADP-ribosyl-[14C]arginine was purified by HPLC
(Hewlett-Packard series 1100 equipped with a diode-array
spectrophotometric detector set at 260 nm) using an anion exchange
perfusion chromatography column (Zorbax Sax 4.6-mm inner diameter × 25 cm, Rockland Technologies, Inc.) and elution with a linear gradient of
NaCl (0-1 M) in 20 mM sodium phosphate buffer,
pH 4.5, for 30 min at a flow rate of 1 ml/min. ADP-ribosyl-[14C]arginine was eluted at 12 min, well
separated from arginine and nicotinamide (which eluted between 2 and 5 min), NAD (16-18 min), and ADP-ribose (40 min).
ADP-ribosyl-[14C]arginine was lyophilized, dissolved in
100 µl of H2O, and stored at
20 °C. Samples (50 ng)
of purified rat brain hydrolase (wild-type or mutants) synthesized as a
GST1 fusion protein were
assayed in 50 mM potassium phosphate, pH 7.5, containing 5 mM dithiothreitol, 10 mM MgCl2, 100 µM ADP-ribosyl-[14C]arginine (78,000 cpm)
(total volume 100 µl). After 1 h at 37 °C, a sample (90 µl)
was applied to a column (0.5 × 4 cm) of Affi-Gel 601 (boronate)
equilibrated and eluted with five 1-ml portions of 0.1 M
glycine, pH 9.0, 0.1 M NaCl, and 10 mM
MgCl2. The total eluate was collected for liquid
scintillation counting. If activity was not detected, the experiment
was repeated with 10 µg of fusion protein and an incubation time of
5 h.
Inhibition of ADP-ribosylarginine Hydrolase--
Samples (50 ng)
of purified rat brain, wild-type hydrolase (synthesized as a GST fusion
protein) in 50 mM potassium phosphate, pH 7.5, containing 5 mM dithiothreitol, 10 mM MgCl2, 100 µM ADP-ribosyl-[14C]arginine (78,000 cpm)
with ADP-ribose (Sigma) as indicated (total volume 100 µl) were
incubated for 1 h at 37 °C. A sample (90 µl) was applied to a
column (0.5 × 4 cm) of Affi-Gel 601 (boronate) equilibrated and
eluted with 0.1 M glycine buffer, pH 9.0, containing 0.1 M NaCl, and 10 mM MgCl2 (five 1-ml
portions). The total eluate was collected for liquid scintillation
counting. Activity is expressed as micromoles of free arginine formed
during the incubation.
ADP-ribose Binding Assay--
[14C]ADP-ribose was
generated in a mixture (300 µl) containing 50 mM
Tris-HCl, pH 7.5, 30 µg of ovalbumin, 20 mM
dithiothreitol, 2 mM (50 µCi) of
[adenine-U-14C]NAD, 100 µM GTP,
5 µg of cardiolipin, 10 mM MgCl2, 50 µg of cholera toxin A subunit, and 50 µg of ADP-ribosylation factor (rARF
1) and incubated for 1 h at 37 °C. After incubation,
[14C]ADP-ribose was purified by HPLC as described for
purification of ADP-ribose-[14C]arginine. To assay
binding of [14C]ADP-ribose, 10 µg of wild-type or
mutant rat hydrolases or bovine serum albumin were incubated (total
volume 100 µl) in 50 mM potassium phosphate, pH 7.5, containing 5 mM dithiothreitol, 10 mM
MgCl2. After 1 h at 37 °C, the mixture was dialyzed
in a Microdialyzer100 (Pierce) for 48 h at 4 °C against
semipermeable membranes (Spectra/Por Membrane MWCO 6-8.000 Spectrum
Medical Industries) in the presence of 0.25 M sucrose, 0.15 M NaCl, 20 mM Tris-HCl, pH 8.0, 5 mM dithiothreitol, 1 mM NaN3, 0.1 mM EDTA. After dialysis, samples and 100 µl from the
dialysis chamber (total volume 100 ml) were counted using a liquid
scintillation counter for calculations of bound and free ADP-ribose.
 |
RESULTS AND DISCUSSION |
The mammalian ADP-ribosylarginine hydrolase gene is, thus far,
unique in the genome. To identify structural features necessary for
activity, evolutionary conservation of sequence was investigated. Deduced amino acid sequences of the mammalian hydrolases contained only
limited regions of identity to the hydrolase from R. rubrum (Table I) (20). Amino acids 60-67,
133-139, and 285-291 of rat, mouse, and human brain hydrolase showed
significant identity with bacterial ADP-ribosylarginine hydrolase.
Conceivably, conserved amino acids in these areas could represent
consensus domains critical for hydrolase function. Of possible
importance with regard to hydrolase function, Asp-60, Asp-61, His-65,
Arg-139, and Asp-285 were deemed possible candidates for active site
residues. His-65 is not present in the R. rubrum enzyme,
where it is replaced by serine.
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Table I
Similarities in deduced amino acid sequences of ADP-ribosylarginine
hydrolases from rat brain and R. rubrum
Differences in amino acid sequences (in single letter code) are noted
by asterisks; a gap is indicated by a dash. Sequences for R. rubrum are from Ref. 20 and those for rat from Ref. 23. In human
hydrolase, leucine 287 is replaced by tyrosine. There are no
differences between rat and mouse hydrolases in these three regions.
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A second domain in the hydrolase, considered potentially crucial in
catalysis, is
Ala-185-X-X-X-X-Gly-Lys-Ser-192
in rat and mouse enzymes, which is a consensus NTP binding sequence. It
differs, however, in human hydrolase, in which -Gly-Lys-Ser-192 is
replaced by -Ser-Arg-Pro-192. Since the enzymes presumably act by
similar mechanisms, this region may not play as important a role in
hydrolase activity as do the corresponding regions in protein kinases
and nucleotide-binding proteins and was not mutated here (26).
Site-directed mutagenesis was used to replace Asp-60, Asp-61, His-65,
Arg-139, and Asp-285 codons with alanine in the ADP-ribosylarginine hydrolase cDNA. The GST-linked fusion proteins (GST-hydrolase) were
purified to homogeneity as described under "Experimental Procedures" (Fig. 1). H65A, R139A, and
D285A mutants had activities similar to that of the wild-type, whereas
the activities of D60A and D61A were three orders of magnitude lower
(Table II). Both D60A and D61A mutant
proteins exhibited the same immunoreactivity as wild-type on Western
blots (data not shown). Activities of WT hydrolase and active mutants,
which were constant for the first 5 h of assay (Fig.
2), were essentially equal and similar to
that of the native rat brain enzyme (24). These data suggest that native and recombinant hydrolase have similar catalytic properties and
that recombinants produced in E. coli can be used as an
appropriate model for the native enzyme. Replacement of Asp-60 or
Asp-61 with Ala, Asn, or Gln resulted in a loss of enzymatic activity.
Mutants D60E and D61E, however, had activity comparable with that of
wild-type (Table III). Double mutants in
which both aspartates were replaced with asparagine, glutamine, or
glutamic acid were inactive (data not shown).

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Fig. 1.
Purification of recombinant
ADP-ribosylarginine hydrolases. ADP-ribosylarginine hydrolases (5 µg), purified on glutathione-Sepharose, were subjected to
electrophoresis in SDS-polyacrylamide gel electrophoresis using 4-20%
gradient gels, followed by staining with 0.05% Coomassie Blue. 1 µg
of bovine serum albumin was loaded as a control. This experiment was
repeated three times with similar results.
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Table II
Specific activities of ADP-ribosylarginine hydrolase constructs: WT,
D60A, D61A, H65A, R139A, and D285A
Purified rat brain hydrolase (as a GST fusion protein) (50 ng) was
assayed in 50 mM potassium phosphate, pH 7.5, 5 mM dithiothreitol, 10 mM MgCl2, 50 µM ADP-ribose-[14C]arginine (39,000 cpm) (total
volume 100 µl). If enzymatic activity was not detected (mutants D60A
and D61A), the experiment was repeated with 10 µg of hydrolase and an
incubation time of 5 h. Specific activity is recorded as in
µmol · min 1 · mg 1 GST fusion
protein. Data are expressed as means of values from three
experiments ± half the range with assays performed in duplicate.
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Fig. 2.
Time course of ADP-ribosylarginine hydrolase
activity of mutants. Purified recombinant rat brain hydrolase (as
a GST fusion protein) (50 ng) was assayed as noted under
"Experimental Procedures." After incubation (1-6 h), a sample was
applied to a column (0.5 × 4 cm) of Affi-Gel 601 (boronate),
equilibrated, and eluted with five 1-ml portions of 0.1 M
glycine, pH 9.0, 0.1 M NaCl, 10 mM
MgCl2. The total eluate was collected for liquid
scintillation counting. Activity of the hydrolases is defined as the
ability to generate free arginine from
ADP-ribosyl-[14C]arginine. Data are means ± S.D. of
values from three experiments, each with assays in duplicate. S.D. bars
within symbols are not shown. , R139A; , D285A; , H65A; ,
WT. Substitution of Ala for His-65, Arg-139, or Asp-285, which are
located in conserved regions, did not change specific activity.
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Table III
Specific activities of ADP-ribosylarginine hydrolase constructs: WT,
D60A, D60E, D60N, D60Q, D61A, D61E, D61N, and D61Q
Experiments were caried out as described for Table II. Specific
activity is recorded as in µmol · min 1 · mg 1 of GST fusion protein. Data are expressed as means of
values from three experiments ± half the range with assays
performed in duplicate.
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Because it was crucial to demonstrate that the catalytically inactive
wild-type and mutant proteins were structurally intact, advantage was
taken of the fact that ADP-ribose (a substrate analogue) is a potent
inhibitor of the enzyme (Fig. 3). GST
fusion proteins were incubated with [14C]ADP-ribose
followed by removal of unbound substrate analogue by dialysis. All
mutants, despite their extreme differences in activity, bound
ADP-ribose in a manner similar to that of the wild-type (Figs. 3 and
4). Affinities of the wild-type and mutant (D60A) ADP-ribosylarginine
hydrolases for ADP-ribose were determined from Scatchard analysis and
were similar (KD = 16 µM in both
instances) (Fig. 4).

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Fig. 3.
Binding of ADP-ribose to hydrolase.
Binding of ADP-ribose to hydrolase was determined as described under
"Experimental Procedures." Data are means of values from duplicate
assays and representative of three for each of the hydrolases. Each
experimental point was determined in three duplicate assays. Mutants
( ) D60A and ( ) D61A, despite their differences in activity, bound
ADP-ribose with an affinity similar to that of the WT enzyme
( ).
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Fig. 4.
Scatchard analysis of ADP-ribose binding to
WT and mutant ADP-ribosylarginine hydrolase D60A. Binding of
[14C] ADP-ribose was quantified as described under
"Experimental Procedures." After incubation for 1 h and
dialysis against semipermeable membranes, hydrolase samples and 100 µl from the dialysis chamber (total volume 100 ml) were counted using
a liquid scintillation counter for calculations of bound and free
ADP-ribose. In the Scatchard plots for wild-type hydrolase
(A) and D60A (B), mean values ± half the
range are given for duplicate assays performed in two separate
experiments. The results are representative of those obtained for D61A
and "double" mutants. All mutants, despite their differences in
activity, bound ADP-ribose with affinity similar to that of the wild
type.
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The concentration of ADP-ribose that decreased enzyme activity by 50%
(IC50) is not equivalent to the KD for
the competitor, but depends on the concentration of the radioligand present in the incubation. For conditions described under
"Experimental Procedures," IC50 = 17 µM
(Fig. 5) and corresponds well to the concentration of ADP-ribose that saturates 50% of the
ADP-ribosylarginine hydrolase under the same conditions (Figs. 3 and
4).

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Fig. 5.
Inhibition of wild-type ADP-ribosylarginine
hydrolase activity by ADP-ribose. Inhibition of hydrolase activity
by ADP-ribose was determined as described under "Experimental
Procedures." Activity of the WT ADP-ribosylarginine hydrolase was
expressed as ability to release free arginine. Data are means of values
from two experiments, each with triplicate assays.
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Catalysis by some hydrolases involves coordinate action of a proton
donor and a nucleophile (26). For ADP-ribosylarginine hydrolase, the
conserved Asp-60 and Asp-61 may be positioned within the proposed
catalytic cleft and could serve as proton donor and nucleophile,
respectively. The positioning of the two aspartic acids would need to
allow sufficient room for an attacking water molecule. In the single
mutants D60E and D61E, sufficient space and flexibility would
accommodate a water molecule, whereas in the double mutant, D60E/D61E,
catalysis could no longer proceed. This study represents the first
identification of critical, active site residues in the
ADP-ribosylarginine hydrolases that appear to be conserved from
bacteria to humans.
 |
ACKNOWLEDGEMENT |
We thank Dr. Martha Vaughan for helpful
discussions and critical review of the manuscript
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Rm. 5N307, Bldg. 10, 10 Center Dr., MSC 1434, Pulmonary-Critical Care Medicine Branch,
NHLBI, National Institutes of Health, Bethesda, MD 20892-1434. Tel.:
301-402-1469; Fax: 301-402-1610; E-mail: konczalp{at}gwgate.nhlbi.nih.gov.
 |
ABBREVIATIONS |
The abbreviations used are:
GST, glutathione
S-transferase;
HPLC, high performance liquid chromatography;
WT, wild-type.
 |
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