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INTRODUCTION |
Penicillamine,
,
-dimethyl-cysteine, is one of the most
unusual amino acids because it is not catabolized by
pyridoxal-5'-phosphate (PLP)-dependent
enzymes.1 Although
penicillamine has the obvious structural similarities to cysteine and
valine, none of the enzymes that degrade Cys or Val can act on
penicillamine. In fact, L-penicillamine can specifically inhibit various PLP-dependent enzymes, whose cofactor is
compelled to form a thiazolidine ring and is no longer bound on the
enzyme (1-4). The driving force for this modification may derive from the unique structure of penicillamine, which bears a hard nucleophile (-NH2) and a soft nucleophile (-SH) on the vicinal carbons.
The
,
-dimethyl group may also help orienting the amino and thiol groups to the same direction and by that promote the thiazolidine ring
formation. D-Penicillamine, in contrast to the
L-isomer, had been considered not to inhibit
PLP-dependent enzymes (5), but later it was shown that the
D-isomer also causes PLP depletion in rat (6, 7).
Myeloperoxidase is another class of enzyme that
D-penicillamine can specifically bind and abolish its
catalytic functions (8, 9). Despite these inhibitory effects,
D-penicillamine has been extensively used in the treatment
of Wilson's disease (10) and in cases of lead poisoning (11). The
curative property is partly due to the ability of
D-penicillamine to bind copper or lead as a stable chelate
that is filterable by the kidney and more importantly due to the
metabolic stability by which penicillamine is not degraded until
excreted in urine. In fact, penicillamine has originally been found in
urine specimens of penicillin-treated patients, suggesting that the
-lactam antibiotic was not degraded beyond penicillamine (12).
Besides these effects on enzymes and metals, penicillamine can exert
various biological effects such as decreasing chemotaxis of
polymorphonuclear leukocytes (13), inhibiting DNA synthesis in T
lymphocyte (14), and inhibiting transactivation of human
immunodeficiency virus , type 1 long terminal repeat (15). Mechanisms
underneath these biological effects are not fully understood yet.
We here report purification and characterization of a novel enzyme
involved in penicillamine degradation in a strain of Bacillus sphaericus that can grow on DL-penicillamine as the
sole nitrogen source. The enzyme, specifically induced when the
bacterial cells were incubated with D- or
L-penicillamine, used NAD and penicillamine as the sole
substrates. The enzyme activity has been assayed by NAD-dependent penicillamine consumption. The enzyme was
purified to the homogeneity using nucleotide affinity ligands and
characterized as a 42-kDa monomeric protein. The catalysis was
irreversible, and penicillamine was derivatized to the phosphoramide
adduct, ADP-penicillamine (Scheme 1). The
phosphoramide product caused potent product inhibition, and a
commercially available Aspergillus adenylate deaminase was
effective in removing the inhibitory product. In the adenylate
deaminase-coupled system, inosine-diphosphate-penicillamine (IDP-penicillamine) was identified as the deaminated form of the product. Kinetic studies on substrate binding interaction and the
product inhibition by ADP-L-penicillamine suggested an
Ordered Bi Bi mechanism with NAD as the first substrate to bind and
ADP-L-penicillamine as the last product to be released. The
enzyme, NAD:penicillamine ADP transferase, showed relaxed
stereospecificity with respect to D- and
L-penicillamine, and the L-isomer was a better
substrate than the D-isomer. The stereochemical preference
in the enzyme reaction was consistent with the observation that the
cells of B. sphaericus consumed L-penicillamine
more rapidly than the D-isomer. The
NAD-dependent modification catalysis may be the first step in the penicillamine degradation pathway in the strain of B. sphaericus.
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EXPERIMENTAL PROCEDURES |
General--
Ultraviolet and visible spectra were measured on
Beckman DU-65 spectrophotometer, and molecular mass was determined by a
quadrupole ion spray mass spectrometer API III (Perkin-Elmer, Canada).
Protein was determined by the method of Bradford using bovine serum
albumin as the standard (16). 1H and 31P NMR
spectra were obtained at 500 and 80.9 MHz using Varian VXR-500 and
VXR-200 spectrometers, respectively, with a Fourier transform accessory
at ambient temperatures.
Screening of Bacteria--
Screening for
penicillamine-catabolizing bacterium has been carried out by the
enrichment culture on a medium containing 0.1% (w/v)
DL-penicillamine, 0.5% glucose, 0.1%
KH2PO4, 0.1% K2HPO4, 0.1% NaCl, 0.05% MgSO4 7H2O, and 0.05% yeast
extract, pH 7.5. Microorganisms were grown at 30 °C under aerobic
conditions, and a bacterium has been isolated as a pale yellow colony
formed on the medium containing 1.3% (w/v) agar.
Enzyme Assays--
Enzyme activity was assayed by measuring the
decrease of penicillamine (method 1) or NAD (method 2). In method 1, the reaction was initiated by adding 10 µl of enzyme solution to 1 ml
of the assay mixture containing 30 mM
L-penicillamine, 30 mM NAD, and 30 mM Tris-HCl buffer, pH 7.5. After incubation at 37 °C
for 15 min, the reaction was stopped by adding 100 µl of 25% (w/v)
trichloroacetic acid. Then an aliquot of 2 µl was spotted on 3MM
chromatography paper (Whatmann) and developed with butanol/acetone/28%
ammonium/water (10:10:5:2). Penicillamine was visualized by spraying
0.5% ninhydrin in 50% aqueous acetone, and the paper was heated at
110 °C for 10 min. The spot of penicillamine was cut out, extracted
in 0.5 ml of 75% ethanol containing 0.05% (w/v) CuSO4,
and determined spectrophotometrically at 510 nm. In method 2, the
amount of NAD remaining in the reaction mixture was determined by the
method of Nussrallar et al. (17). The sample was diluted to
contain less than 0.3 mM of NAD, and the 100-µl aliquot
was mixed with 100 µl of ethanol and 700 µl of 0.1 M
glycine/NaOH buffer, pH 10.0. Equine liver alcohol dehydrogenase (0.1 units/100 µl) was added to the solution, and the absorbance at 340 nm
was monitored for 3 min. The slope of the time-dependent
increase in the absorbance was used to estimate the concentration of NAD.
Preparation of Crude Cell Extract--
B. sphaericus
was grown on 4 liters of Luria-Bertani broth for 16 h at 37 °C
and then inoculated to 30 liters of the same medium in a 40-liter
vessel fermentor equipped with a mechanical stirrer. Cells were grown
under aerobic conditions at 37 °C until an early stationary phase,
harvested by centrifugation at 7,000 × g for 15 min
and then washed twice with cold 20 mM Tris-HCl buffer, pH
7.5. The cells (wet weight, 120 g) were suspended in 2 liters of
the Tris buffer, pH 7.5, and incubated at 30 °C for 6 h, and
then DL-penicillamine (1% w/v) was added and incubated for
another 6 h. Cells were suspended in 500 ml of the same buffer containing phenylmethylsulfonyl fluoride (0.1 mM) and
disrupted by sonication for 20 min, and cell debris was removed by
centrifugation. Ammonium sulfate was added to 80% of the saturation,
and precipitates were dissolved in the buffer (50 ml, pH 7.5) and
dialyzed twice against 2 liters of the same buffer. Insoluble materials
were removed by centrifugation, and the supernatant solution was used as the crude cell extract of B. sphaericus.
Enzyme Purification--
Enzyme purification was carried out at
under 4 °C, and 20 mM Tris-HCl buffer, pH 7.5, containing 0.01% 2-mercaptoethanol (buffer A) was used throughout the
procedure. The crude cell extract was applied to DEAE Toyopearl 650 M column (5.0 × 30 cm) equilibrated with buffer A. After washing the column extensively with buffer A, the enzyme activity
was eluted with a 0-0.5 M KCl linear gradient in the
buffer. Active fractions were concentrated on Amicon YM-10 membrane and
applied to a DyeMatrex Blue B column (15 mm × 14 cm), which was
equilibrated with buffer A. The column was washed with 2 volumes of 1 mM NADH and 1 mM NAD in the same buffer, and the enzyme was eluted with a linear gradient of KCl (0-0.5
M) contained in buffer A. Active fractions were combined
and concentrated on Centricut-mini (Kurabou, Japan). The enzyme was
absorbed on the Dyematrex Orange A column equilibrated with buffer A. The column was washed with 1 mM of NADH and 1 mM of NAD in the buffer, and the enzyme was eluted with a
0-0.5 M KCl linear gradient. Active fractions were
concentrated, and applied to TSK-GEL 3000SW column (7.5 × 600 mm,
TOSOH, Japan) equilibrated with buffer A containing 0.1 M
KCl, and the active fractions were combined and concentrated on
Centricut-mini.
SDS-Polyacrylamide Gel Electrophoresis and Gel
Filtration--
Molecular mass of the enzyme was determined by
SDS-polyacrylamide gel electrophoresis according to the method of
Laemmli (18). Purified enzyme was applied to a TSK-GEL 3000SW column in
0.1 M sodium phosphate buffer, pH 7.5, containing 0.1 M sodium sulfate to estimate the native molecular size. The
following molecular markers were used to calibrate the column:
myoglobin (17,000), chicken ovalbumin (44,000), bovine gamma globulin
(158,000), and bovine thyroglobulin (670,000).
Analytical HPLC--
Substrate disappearance and product
formation were monitored on reverse-phase high performance liquid
chromatography (HPLC). After protein was removed from the reaction
mixture (1 ml) by adding 250 µl of 25% trichloroacetic acid and
subsequent centrifugation, a 20-µl aliquot of the sample was injected
onto reverse-phase HPLC system consisting of Waters 600E system
controller, an on-line degasser, and Waters 484 Tunable Absorbance
Detector. Reverse-phase separation was achieved on µBondasphere C18
column (19 mm × 15 cm) using a linear gradient of 0-5%
acetonitrile in 50 mM sodium acetate buffer, pH 3.5, at a
flow rate of 5.0 ml/min. Eluate was monitored by the absorbance at 260 nm.
Purification of Enzyme Reaction Product--
A reaction mixture
(5 ml, pH 7.5) containing 30 mM NAD, 30 mM
L-penicillamine, adenylate deaminase (10 units; Sigma), and the purified enzyme (10 units) was incubated at 37 °C for 6 h. Enzyme reaction product has been purified by four successive
preparative HPLC systems. 1) Preparative HPLC was performed as
described under "Analytical HPLC." 2) HPLC employed Superdex
Peptide HR column (10 × 300 mm) eluted with deionized water at a
flow rate of 0.25 ml/min. 3) Preparative HPLC was again performed on
µBondasphere C18 column with 0-35% linear gradient of methanol in
deionized water at a flow rate of 2.5 ml/min. 4) Ion exchange HPLC on a TSK-GEL DEAE-5PW column (7.5 × 75 mm) has been performed with a
linear gradient of 10-300 mM NH4HCO3 buffer (pH 8.2).
Eluate was monitored at 260 nm, and concentrated by lyophilization at each step of the preparative HPLC.
Chemical Synthesis of
ADP-L-Penicillamine--
ADP-L-penicillamine
has been chemically synthesized by the method described by Rossomondo
et al. (20) with some modifications, and 31P NMR
spectra of reaction mixture were obtained at each step of the
synthesis. To the aqueous solution of ADP (5 mmol/8 ml), which was
adjusted to pH 8 with triethylamine,
1-ethyl-3-(3dimethylaminopropyl)-carbodiimide hydrochloride (500 mmol/2
ml water) was added dropwise and stirred at room temperature for 30 min. An aqueous solution of L-penicillamine (50 mmol/10 ml,
pH 8, with triethylamine) was added to the mixture, and the solution
was incubated at 50 °C for 2 h. The reaction mixture was passed
through a column (0.7 × 5 cm) of activated charcoal (Wako,
Japan), and the column was thoroughly washed with deionized water.
ADP-L-penicillamine was eluted with 50% aqueous ethanol,
and was further purified by the preparative HPLC described above in 4).
Purity of the synthetic ADP-L-penicillamine was verified by
analytical reverse-phase HPLC.
Kinetic Studies--
Kinetic properties of NAD:penicillamine ADP
transferase were examined using the purified enzyme, and the activity
was assayed by method 1. For kinetic studies, adenylate deaminase was
not included in the assay mixture. Initial velocities of penicillamine disappearance were measured during the first 15 min, where we observed
linear time-dependent substrate disappearance. Data were obtained by varying the concentration of one substrate with the fixed
concentrations of the other, and presented as double-reciprocal plots
as initial velocity versus varied substrate concentrations. Product inhibition studies were performed by including chemically synthesized ADP-L-penicillamine in the assay mixture at
several fixed concentrations and varying the concentration of either
L-penicillamine or NAD in the presence of fixed
concentration of the other substrate (30 mM
L-penicillamine or 30 mM NAD).
Double-reciprocal plots using initial velocity versus varied
substrate concentrations were used to determine the mode of inhibition
(21).
 |
RESULTS |
Penicillamine-catabolizing Bacterium--
A
penicillamine-catabolizing bacterium has been isolated from a local
soil sample collected in our university campus. The bacterium was
identified as a strain of B. sphaericus based on the
following biological characteristics: the microorganism was a
Gram-positive, spherical spore forming, catalase positive bacterium, and it can grown on media at pH 6.0 but not on media containing 10%
(w/v) NaCl. Starch was not used as a carbon source.
Enzyme Induction--
The bacterium did not show the ability to
consume DL-penicillamine when grown on Luria-Bertani broth.
The enzyme activity was most effectively induced when the cells were
incubated in 20 mM Tris-HCl buffer, pH 7.5, at 30 °C for
6 h and then further incubated in the presence of 0.1% (w/v)
DL-penicillamine for another 6 h. Cells treated with
0.1% DL-penicillamine consumed L-penicillamine at the rate of 0.3 µmol/min/g of wet cells at 30 °C. The rate of
D-penicillamine uptake was less than 0.1 µmol/min under
the same conditions. The enzyme was induced by D- or
L-penicillamine, and in either case cells consumed
L-penicillamine more rapidly than the D-isomer.
L-Valine, L-cysteine, L-leucine,
and L-isoleucine did not induce the activity in the
bacterial cells.
Penicillamine Consumption in Crude Cell
Extract--
Penicillamine-consuming activity in the cell extract has
diminished when small molecules were removed by dialysis. To identify the molecules required for the activity, we have incubated the cell
extract with 30 mM of NAD, NADP, NADH, NADPH, PLP, FAD, and FMN, respectively, with 30 mM DL-penicillamine,
and penicillamine disappearance was measured by ninhydrin. Among the
compounds tested, only NAD promoted the penicillamine consumption. When
NAD and DL-penicillamine were incubated with cell extract
for 1 h, NAD decreased at the rate of 1.5 ± 0.1 µmol/min,
whereas penicillamine decreased at the similar rate of 1.6 ± 0.1 µmol/min. The amount of NAD remained unchanged when
DL-penicillamine was omitted (Fig. 1A). The activity was not
detected when the cell extract had been boiled for 10 min. Thus, the
enzymatic penicillamine consumption appeared to be a
NAD-dependent catalysis. Interestingly, we could not detect
the formation of NADH by the UV absorbance at 340 nm, although 9 mM of NAD disappeared in the incubation period of 60 min.
The concentration of thiol group in the crude cell extract, simultaneously determined by 5,5'-dithiobis-(2-nitrobenzoate), also
decreased but not in the accordance with the disappearance of amino
group (Fig. 1A, inset). Despite the competent
enzyme activity induced in the cell extract, our preliminary attempts to identify the enzyme reaction products were not successful due to the
small yield of products. It seemed likely that crude cell extract of
B. sphaericus contained enzymes for the succeeding pathway,
and therefore the products were not accumulated.

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Fig. 1.
NAD-dependent penicillamine
consumption. A, crude cell extract of B. sphaericus (80 mg protein/10 ml) was incubated with 30 mM DL-penicillamine and 30 mM NAD,
at pH 7.5, and the decrease of DL-penicillamine ( ) and
NAD ( ) was monitored. The concentration of NAD ( ) remained
unchanged until 30 mM DL-penicillamine was
added as indicated by the arrow. The inset shows
the time course of decrease in amino and thiol groups in the reaction
mixture. Timed aliquots (20 µl) were reduced with 50 µg/ml NaBH4,
mixed with 10 µl of acetone, and then assayed for amino group ( )
by ninhydrin reagent and thiol group ( ) by
5,5'-dithiobis-(2-nitrobenzoate). B, purified enzyme (12 µg; 10 units) was incubated with 30 mM
L-penicillamine and 30 mM NAD at pH 7.5 in the
absence ( ) and presence ( ) of Aspergillus adenylate
deaminase (10 units).
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Enzyme Purification and Characterization--
The
NAD-dependent penicillamine-consuming enzyme was purified
to the homogeneity with a yield of 7.7% in the four-step procedure summarized in Table I. Nucleotide
affinity ligands, DyeMatrex Blue B and DyeMatrex Orange A were
efficient means for this enzyme. Other affinity ligands such as
DyeMatrex Blue A, Red A, and Green A were not effective for this
enzyme. The purified enzyme showed the specific activity of 850 units/mg of protein. Gel filtration chromatography on TSK-GEL 3000SW
column was used to estimate the molecular weight of the native enzyme
as 42,000, and SDS-polyacrylamide gel electrophoresis analysis
suggested that the denatured protein has the molecular mass of 42 kDa
(Fig. 2). The purified enzyme did not
have chromophore groups detectable in the ultraviolet and visible
regions. The enzyme showed high substrate specificity to penicillamine
and NAD. Analogous amino acids such as L-valine, L-cysteine, L-cystine,
L-homocysteine, L-leucine, and
L-isoleucine did not serve as a substrate.
D-Penicillamine was a poor substrate with the
Km value of 200 mM. Nucleotides such as
NADH, NADP, NADPH, ATP, ADP, AMP, ADP-ribose, nicotinamide
mononucleotide, and nicotinamide did not substitute for NAD. Nucleotide
triphosphates such as GTP, CTP, and TTP were also inert as the
substrate. The effect of pH was examined using 50 mM
potassium phosphate buffer (pH 5.5-7.5) and 50 mM Tris-HCl
buffer (pH 7.0-8.5). The optimum pH was in a narrow range around 7.5;
at pH 6.5 and 8.0 the activity decreased to 25 and 40% of the optimum
activity, respectively. Although the choice of buffer was not as
significant as the pH values, the activity with the phosphate buffer
was about 85% of the activity with Tris buffer at the optimal pH 7.5. The optimum temperature for the catalysis was 37 °C.

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Fig. 2.
SDS-polyacrylamide gel electrophoresis of the
purified NAD:penicillamine ADP transferase from B. sphaericus. Lane 1, marker proteins: lane
2, 5 µg of purified enzyme.
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Preparation of Enzyme Reaction Product--
Potent product
inhibition has been observed when the purified enzyme (12 µg; 10 units) was incubated for more than 1 h with 30 mM
L-penicillamine and 30 mM NAD at pH 7.5. The
rate of substrate disappearance decreased in 1 h, and only 7% of
L-penicillamine was used in 6 h (Fig. 1B).
Supplementing enzyme to the mixture did not restore the activity. We
tested ADP, ADP-ribose, AMP, and nicotinamide mononucleotide to see
whether they could be inhibitory to the purified enzyme. ADP was found
to act as a potent inhibitor (IC50 = 20 µM),
whereas other compounds had no effects. To circumvent the product
inhibition we tested adenylate deaminase and adenylate kinase to see if
either of them could restore the catalysis. Adenylate kinase had no
effects, but in the presence of adenylate deaminase (10 units),
penicillamine consumption continued for 6 h, and the concentration
of L-penicillamine has decreased to 6 mM (Fig.
1B). The reaction mixture was analyzed on the analytical
reverse-phase HPLC system, and the eluate was monitored by the
absorbance at 260 nm. Besides the remaining substrate NAD (24.0 min),
nicotinamide (27.1 min), and an unknown compound (16.3 min) were
detected in the reaction mixture. The unknown product was purified by
the four-step preparative HPLC and lyophilized to give 75 mg of white powder. 1H NMR (500 MHz: D2O):
1.33 (3H, s), 1.43 (3H,
s), 3.62 (1H, s), 4.02 (2H, m), 4.38 (2H, m), 6.03 (1H, d, J = 5.9 Hz), 8.10 (1H, s), 8.37 (1H, s). 31P NMR (80.9 MHz: D2O):
- 8.9 (d, J = 21 Hz), 0.79 (dt, J = 21 Hz, 9.8 Hz).
Characterization of the Enzyme Reaction Product--
The enzyme
reaction product did not react with ninhydrin, but acid-catalyzed
hydrolysis of the product liberated penicillamine molecule, which was
identified as a ninhydrin-positive spot on thin layer chromatography.
Other chemical tests indicated the presence of thiol group (by platinum
chloride-potassium iodide reagent), phosphate (by ammonium
molybdate-perchloric acid reagent), and ribose (by orcinol-iron (III)
chloride-sulfuric acid reagent) contained in the compound.
1H NMR showed the signals assigned to
,
-dimethyl
group (
1.33 and
1.43) and
-proton (
3.62) of
penicillamine, and the protons for ribose at 4.02, 4.38, and 6.03 ppm.
Singlet signals at
7.8 and
8.2 indicated the presence of a
purine base, which was identified as inosine by the absorption maximum
at 248.5 nm. Fiske-SubaRow assay (22) showed that the product contained
two phosphates for one inosine base, which was determined by the UV
absorption at 248.5 nm (
= 12.2 mM
1).
These results suggest that the product may consist of penicillamine and
IDP. Besides the result that the compound was ninhydrin-negative, several lines of evidence suggested that the amino group of
penicillamine and IDP are bound by a phosphoramide linkage. First,
alkaline phosphatase and 5'-nucleotidase failed to release inorganic
phosphate from the product, suggesting that the
-phosphate of the
IDP group was occupied. Secondly, the enzyme reaction product was
stable in alkaline solution but labile in acidic conditions.
Phosphoramide bond has been reported to be labile in acidic solution
but stable in alkaline solution (23), whereas esters and acyl phosphate are unstable in alkaline solution (24). The enzyme reaction product has
decomposed to IDP and penicillamine under the acidic conditions, but it
survived the alkaline conditions (Fig.
3). The decomposition reaction was
reproducible but not reversible. The 31P NMR spectrum of
the enzyme reaction product revealed two signals of the same peak
intensity at
8.9 (d, J = 21 Hz) and
0.79 (dt, J = 21, 9.8 Hz) (Fig. 4). Quadrupole ion
spray mass spectrometry showed that the product had the molecular mass
of 558.5 in the negative ion mode, and it was in good agreement with
the calculated mass for IDP-penicillamine, [M-H]
= 558.3735. Thus, the product was identified as IDP-penicillamine, the
phosphoramide compound linked between the amino group of penicillamine and
-phosphate of IDP. Nicotinamide-ribose, the other product that
would derive from NAD, was not detected in the reaction mixture, but
nicotinamide has been identified in the reaction mixture. We have
observed that nicotinamide-ribose prepared in situ by incubating nicotinamide mononucleotide with calf intestine alkaline phosphatase at pH 7.5 spontaneously decomposed to nicotinamide and
ribose (data not shown).

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Fig. 3.
Stability of the enzyme reaction product in
acidic/alkaline aqueous solutions. The enzyme reaction product was
incubated at pH 12.0 (A) or 2.0 (B) at 37 °C
for 12 h, neutralized, and analyzed on DEAE-5PW column with linear
gradient of 10-300 mM NH4HCO3. The eluate was monitored at
210 and 260 nm. Penicillamine (10.5 min) and IDP (20.7 min) have been
identified by co-chromatography with the authentic compounds.
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Fig. 4.
31P NMR of the enzyme reaction
product, IDP-penicillamine. The enzyme reaction product (10 mg)
was dissolved in 700 µl of D2O, and 31P NMR
spectrum was collected at 80.9 Hz at an ambient temperature. The
spectrum is an average of 300 acquisitions and is referenced to an
external standard of 85% phosphoric acid as 0 ppm.
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Chemical Synthesis of
ADP-L-Penicillamine--
ADP-L-penicillamine
has been chemically synthesized from ADP and
L-penicillamine, and the reaction mixture was observed on 31P NMR spectrometry at each step of the synthesis. On
proton-decouple 31P NMR, ADP gave two doublet signals,
-phosphate at
8.9 (d, J = 21 Hz) and
-phosphate at
4.9 (d, J = 21 Hz). Then, the
-phosphate, activated by
1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide hydrochloride, has
shifted upfield by 13 ppm. Upon addition of L-penicillamine, the
-phosphate signal has appeared at
0.79 as the double triplet signal. The
-phosphate signal at
8.9 did not change throughout the synthesis. Purified
ADP-L-penicillamine showed the two signals of the same peak
intensity at
8.9 (d, 21 Hz) and
0.79 (dt, 21, 9.8 Hz).
Kinetic Studies--
Double-reciprocal plots gave converging lines
when L-penicillamine was the variable substrate at fixed
concentrations of NAD (Fig.
5A). The same pattern was also
observed when NAD was the variable substrate using fixed concentrations
of L-penicillamine (Fig. 5B). A replot of the
intercepts against the reciprocal of the concentration of fixed
substrate was extrapolated to the x axis to reveal the
Km values for each substrate (Fig. 5, A
and B, right graphs). Km
values for L-penicillamine and NAD were 6.5 and 13.0 mM, respectively. Product inhibition studies were performed
by including chemically synthesized ADP-L-penicillamine in
the assay mixture. When L-penicillamine was the variable
substrate in the presence of 30 mM NAD, the
double-reciprocal plot of the initial velocity data revealed a pattern
consistent with noncompetitive inhibition (Fig.
6A). When NAD was the variable
substrate, the double-reciprocal plot revealed a pattern consistent
with competitive inhibition (Fig. 6B). As calculated from
replots on the Dixon plot (Fig. 6B, inset), the
Ki value of ADP-L-penicillamine was 20 µM.

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Fig. 5.
Initial velocity patterns with NAD and
L-penicillamine as the substrates. The reaction was
initiated by adding 20 µg of the purified enzyme to the reaction
mixture, and incubated for 15 min at 37 °C. A, the
concentration of L-penicillamine was varied at the fixed
concentrations of NAD at 15 ( ), 30 ( ), 45 ( ), and 60 ( )
mM. B, the concentration of NAD was varied at
the fixed concentrations of L-penicillamine at 5 ( ), 10 ( ) 15 ( ), and 30 ( ) mM.
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Fig. 6.
Product inhibition by
ADP-L-penicillamine. A,
ADP-L-penicillamine was included in the assay mixture at
the concentration of 2 ( ), 20 ( ), 50 ( ), and 80 ( )
µM. L-Penicillamine was the variable
substrate, and the concentration of NAD was fixed at 30 mM.
B, the concentration of ADP-L-penicillamine was
20 ( ), 32.5 ( ), 60 ( ), and 90 ( ) µM. NAD was
the variable substrate, and the concentration of
L-penicillamine was fixed at 30 mM. The
inset represents the Dixon plot to compute the
Ki value of ADP-L-penicillamine. The
concentration of NAD was set at 30 ( ), 60 ( ), and 90 ( )
mM, and the intersect at the x axis revealed the
Ki values of 20 µM.
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DISCUSSION |
Amino acids, when utilized as a nitrogen source, are usually
catabolized by PLP-dependent transamination or by
NAD-dependent oxidative deamination. We have demonstrated
in this paper that the penicillamine degradation in B. sphaericus may be initiated by a novel NAD-dependent
modification catalysis that yields ADP-penicillamine, a phosphoramide
compound linked between
-phosphate of ADP and the amino group of
penicillamine. The existence of biologically relevant phosphoramide
compounds has been documented for N-phosphor-creatine formation from ATP and creatine, AMP phosphoramide synthesis from ATP
and ammonium in Mycobacterium avium (25), and the
heptapeptide antibiotic microcin C7, which contains O
-(3-aminopropanol)-AMP group linked to the C terminus carbonamide (26).
In addition to these phosphoramide compounds, it has been demonstrated
that Escherichia coli DNA ligase and T4 bacteriophage DNA
ligase form a covalently bound enzyme-AMP intermediate in which a lysyl
-amino group and AMP are linked by phosphoramide bond (27). E. coli ligase uses NAD as the substrate for the enzyme-AMP
intermediate formation, whereas the bacteriophage enzyme uses ATP as
the substrate. The enzyme reaction product elucidated in the present
study differed from these known phosphoramides in that the amide bond
is formed on the pyrophosphate group of ADP, and thus the enzyme
reaction represents a new class of NAD-dependent
modification catalysis. The novel enzyme, which showed high substrate
specificity to NAD and penicillamine, has been termed as
NAD:penicillamine ADP transferase.
The enzyme reaction product, IDP-penicillamine, has been prepared in
the adenylate deaminase-coupled system and characterized by use of
1H and 31P NMR spectrometry, mass spectrometry,
and other chemical tests. The phosphoramide bond between IDP and
penicillamine has been suggested by the result that the product was
stable in alkaline solution but labile in acidic solution, in which the
product irreversibly decomposed into IDP and penicillamine. The
molecular mass determined by quadrupole ion spray mass spectrometry
agreed with that of IDP-penicillamine. Proton-decouple 31P
NMR spectrum showed two signals at
8.9 and
0.79. The doublet signal at
8.9 was assigned to the
-phosphate of
IDP-penicillamine, representing the geminal homonuclear coupling with
the
-phosphate by 21 Hz. Although the double-triplet signal at
0.79 has been assigned to the
-phosphate based on the comparison
with 31P NMR spectra of chemically synthesized
ADP-penicillamine, we could not give a clear explanation for the 9.8-Hz
splitting besides the geminal coupling by 21 Hz. As one of likely
reasons, we consider that this additional splitting may be representing
the equilibrium of distinct conformational states caused by restricted
pseudo-rotation on the
-phosphate.
Enzyme purification has been performed by following the activity of
NAD-dependent L-penicillamine consumption. We
could not obtain homogeneously purified enzyme by conventional
procedure using DEAE Toyopearl, Butyl Toyopearl and Mono Q (data not
shown). The enzyme has been successfully purified when affinity ligands DyeMatrex Blue B and Orange A were employed in the purification. Physicochemical properties, substrate specificity, and kinetic properties were examined using the purified enzyme. Because
nicotinamide-ribose spontaneously hydrolyzed to nicotinamide and
ribose, the NAD-dependent modification catalysis appeared
to be an irreversible process. Therefore, the application of kinetic
analysis was limited to initial velocity studies of the forward
reaction in the absence and presence of the inhibitory product
ADP-L-penicillamine. The substrate interaction studies and
product inhibition kinetics were indicative of an Ordered Bi Bi
mechanism, and product inhibition kinetics were used to determine the
order of substrate binding and product release. NAD appeared to be the
first substrate to bind and ADP-L-penicillamine is the last
product to be released. According to the Dixon plot on the competitive
inhibition, ADP-L-penicillamine showed much higher affinity
to the enzyme (Ki = 20 µM) than the
substrates, NAD (Km = 13.0 mM) and
L-penicillamine (Km = 6.5 mM).
The high Km values for NAD and
L-penicillamine have given rise to the question of whether
they are the true substrates for this novel transferase. Although our
efforts of substrate screening were within a limit of commercial
availability, the purified enzyme showed exclusive substrate
specificity among the compounds tested. L-Valine,
L-cysteine, L-cystine,
L-homocysteine, L-leucine, and
L-isoleucine were inert substrates, and
D-penicillamine served as a substrate with only 30-fold
lower reactivity compared with the L-isomer. Thus, the
enzyme appears to recognize the set of bulky substituents of
,
-dimethyl and thiol groups, which constitute the unique
structure of penicillamine. The observation that the enzyme was
specifically induced by L- or D-penicillamine provides more compelling evidence that the enzyme is biologically relevant to the penicillamine metabolism in the strain of B. sphaericus. As for the other substrate that donates ADP to
penicillamine, we also tested various mononucleotides and
dinucleotides, and only NAD served as the substrate. The observation
that NADP, NADPH, and NADH did not substitute NAD also indicated the
exclusive substrate specificity to NAD. Although we cannot completely
rule out the possibility that an unidentified ADP donor with a smaller
Km value might be synthesized in the bacterial
cells, it would be more convenient for the cells to use NAD as the
substrate for penicillamine modification. It is conceivable in this
case that the high Km value helps to prevent
unnecessary NAD consumption. It has been reported that enzymes such as
glutamate dehydrogenase (28), alanine aminotransferase (29), and
glycine aminotransferase (19) show very high substrate specificity, but
their apparent Km values are high in a range of
several millimolar levels. In such cases, however, the
Km values are compensated by succeeding enzyme
reactions that efficiently consume the product and thus draw the
equilibrium to the forward reaction. The contribution of succeeding
catalysis would be especially important for NAD:penicillamine ADP
transferase because its phosphoramide product
ADP-L-penicillamine causes potent inhibition on the enzyme.
In the present study, we used a commercially available adenylate
deaminase to prevent the product inhibition by converting ADP-L-penicillamine to IDP-L-penicillamine.
Although the enzyme was effective in removing the inhibitory product,
we could not detect the corresponding activity in the cell of B. sphaericus. The penicillamine degradation pathway in the bacterium
probably employs a different type of catalysis for removing
ADP-L-penicillamine. This hypothesis may be substantiated
by the observation that the thiol group slowly decreased in the cell
extract (Fig. 1, inset). The catalysis of NAD:penicillamine
ADP transferase leaves the thiol group of penicillamine unchanged, and
therefore the time-dependent thiol disappearance strongly
suggests that the thiol group of ADP-penicillamine would be removed in
the succeeding pathway.
Although the novel NAD-dependent modification catalysis did
not give us any clue on how penicillamine can be degraded and how the
amino group of penicillamine is used as the nitrogen source by B. sphaericus, the enzyme reaction may be the first, rate-limiting step in the penicillamine degradation in the bacterial cell. Specific enzyme induction by D- or L-penicillamine and
exclusive substrate specificity suggest that the enzyme would be
involved in the penicillamine assimilation pathway.
L-Penicillamine was a better substrate than the
D-isomer, and this was consistent with the observation that the bacterial cells consumed L-penicillamine more rapidly
than the D-isomer. Vigorous penicillamine consumption
observed for crude cell extract, in contrast to the potent product
inhibition on the purified enzyme, may be explained if the crude
extract contained the enzymes that catalyze succeeding reactions. The succeeding enzyme reactions are still under our investigation on the
premise that the thiol group should be the next target to be degraded.
Considering the reaction sequence by which PLP-dependent enzymes are inactivated by penicillamine, modification of the amino
group may also serve to protect PLP-dependent enzymes in the cell of B. sphaericus.