460 植物生理与分子生物学学报
Some Properties of the Allantoate Amidohydrolase from French Bean
Seedlings
XU Zhi-Wei1*, ZHOU Hua-Xin2, HUANG Wei-Nan2
(1Department of Internal Medicine, University of Iowa, Iowa, IA52242, USA; 2 Fujian Institute of Subtropical Botany, Xiamen 361006, China)
Abstract: Allantoate degradation was demonstrated
in the extracts of ungerminated seeds and roots, stems
and leaves in germinated seedlings of French bean
(Phaseolus vulgaris L.). Activity of allantoate-degrad-
ing enzyme could only be measured when phenylhy-
drazine was included in the assay mixture. Partial pu-
rification of allantoate-degrading enzyme from seed-
lings was performed and two fractions with allantoate-
degrading enzyme activity were obtained. The molecu-
lar mass of the first fraction was over 200 kD and that
of the second one was 13.5 kD. The allantoate-degrad-
ing enzyme with small molecular weight contained no
activity of either ureidoglycolate-degrading enzyme or
urease. From the stoichiometry of the reaction cata-
lyzed by the allantoate-degrading enzyme with small
molecular weight it followed that the enzyme was
allantoate amidohydrolase (EC 3.5.3.9). The optimal
pH for the allantoate amidohydrolase was 8.5. Mn2+
ions were essential for enzymatic activity. Glyoxylate
and glycolate strongly inhibited the enzyme activity.
The lysine and tryptophan residues were essential to
the enzymatic catalysis; thiol group and tyrosyl resi-
dues were not involved in the enzyme catalysis.
Key words: allantoate amidohydrolase; properties; French bean
Ureides (allantoin and allantoate) are the major
nitrogen compounds in xylem sap of a variety of ni-
trogen-fixing plants, including several important le-
gumes such as soybeans, French bean (Thomas and
Schrader 1981, Reynolds et al. 1982, Schubert 1986,
Atkins et al. 1992). The hydrolysis of allantoin to
allantoate by allantoinase (allantoin amidohydrolase,
EC 3.5.2.5) in plants is well documented (Wells and
Lees 1992, Bell and Webb 1995), but for a long time
there has been uncertainty about the pathway of
allantoate breakdown in higher plants. Two enzymes
30 卷
catalyting allantoate hydrolysis are known: allantoate
amidinohydrolase (allantoicase, EC 3.5.3.4) and
a l l a nt oa t e a mi do hyd r o l a s e [ a l l a nt oa t e
amidinohydrolase (decarboxylating), ALAH, EC 3.5.
3.9] (Xu et al. 1995). The first enzyme catalyzes the
hydrolysis of 1 mol of allantoate to 1 mol of
ureidoglycolate and 1 mol of urea, while the reaction
ca ta l yz e d b y ALAH gi ves ri se to 1 mol of
ureidoglycolate, 1 mol of CO2 and 2 mol of ammonia
(Vogels and van der Drift 1976).
Allantoicase was found in bacteria grown aero-
bically on allantoin, e.g. Pseudomonas species,
Arthrobacter globiformis, Candida utilis and fungi
Aspergillus nidulans (Vogels and van der Drift 1976),
Burkholderia cepacia (Lowe et al. 2001), yeast Sac-
charomyces cerevisiae (Yoo and Cooper 1991), algae
Chlamydomonas reinhardtii (Piedras et al. 2000) and
animals (Vigetti et al. 2002). ALAH was shown to be
present in bacteria growing on allantoin anaerobically,
e.g. Streptococcus allantoicus (van der Drift and Vogels
1969a), Escherichia coli and Proteus rettgeri (Vogels
1966, Vogels and Van der Drift 1976), or aerobically,
e.g. Bacillus fastidiosus (Xu et al. 1995), Bacillus
subtilis (Schultz e t al. 2001), Pse udomonas
acidovorans (Vogels and van der Drift 1976). Although
evidence had been presented (Shelp and Ireland 1985)
that soybean contained allantoicase, subsequent experi-
ments suggested that the enzyme responsible for
allantoate hydrolysis was ALAH (Winkler et al. 1987,
Lukaszewski et al. 1992). However, these results were
Received 2003-12-30, Accepted 2004-05-08.
This work was supported by the Natural Science Foundation of Fujian
Province (No.B9910026).
*E-mail: zhiwei-xu@uiowa.edu
Abbreviation ADE: allantoate-degrading enzyme; ALAH: allantoate
amidohydrolase.
4期 徐志伟等: 四季豆幼苗中尿囊酸酰胺水解酶的一些特性(英文)
obtained with whole plants and crude extracts and so
fa r a lla ntoa te-degra ding enz yme (a lla ntoa te
amidohydrolase or allantoate amidinohydrolase) has
not been partially purified from higher plant, due to
the method problem for the enzyme assay. Purifica-
tion and characterization of allantoate amidohydrolase
will be needed to fully understand the pathway of ure-
ide ca t a bolism (Winkler e t al. 19 88a ). The
ureidoglycolate formed as a result of allantoate break-
down in pla nt is further degra ded either by
ureidoglycolate amidohydrolase (EC 3.5.3.19) in
French bean (Wells and Lees 1991, Xu et al. 1999),
which hydrolyzes ureidoglycolate to glyoxylate, CO2
and ammonia or by ureidoglycolate urea-lyase
(ureidoglycolase, EC 4.3.2.3) in the legume chickpea
(Munoz et al. 2001) to glyoxylate and urea.
This paper describes for the first time the mea-
surement of allantoate hydrolysis and reports the par-
tial purification and properties of ALAH from French
bean seedlings.
1 Materials and Methods
1.1 Plant material
French bean seeds (Phaseolus vulgaris L. cv.
baishui) were planted in vermiculite and incubated at
25℃ under a 12-h period of daylight per day. After 6-
8 d the seedlings, with the exclusion of cotyledons,
were collected and stored at -30℃.
1.2 Preparation of crude extract
Plant tissue was cut into pieces and homogenized
with a blender in 3 volumes of Tris-HCl buffer (50
mmol / L, pH 7 . 5 ) c ont a i ni ng 1 % ( W / V )
polyvinylpyrrolidone. The homogenate was passed
through four layers of cheese cloth and then centri-
fuged at 38 000×g for 30 min at 4℃. The supernatant
was used as crude enzyme preparation.
1.3 Partial purification of allantoate-degrading
enzyme
Crude enzyme was extracted from seedlings with-
out cotyledons (800 g) with Tris-HCl buffer (50 mmol/
L, pH 7.5) containing 0.1 mmol/L EDTA and 1% poly-
vinylpyrrolidone as described above. All purification
steps were carried out at 4℃. The crude extract was
brought to 25% saturation with ammonium sulfate by
461
stepwise addition of the salt under continuous gentle
stirring. After 30 min at 4℃ the suspension was cen-
trifuged at 18 000×g for 30 min. The supernatant was
saturated to 50% ammonium sulfate, stirred and cen-
trifuged as described above. The resulting pellet was
resuspended in Tris-HCl buffer (50 mmol/L, pH 7.5)
containing 0.1 mmol/L EDTA (buffer A) and used for
gel filtration. The protein solution was applied to a
Sephacryl S-200 column (1.6 cm×97 cm, Pharmacia)
equilibrated with buffer A. Protein was eluted with
buffer A at a flow rate of 41.5 mL/h. Two activity peaks
with allantoate-degrading activity were found after 82
mL and 183 mL of elution were passed, and were
refered as ADE1 and ADE2 respectively. The fractions
containing the two peaks were pooled separately.
The ADE1 activity pool was applied to a DEAE-
Sephadex A50 column (2.6 cm×25 cm, Pharmacia)
equilibrated with Tris-HCl buffer (50 mmol/L, pH 7.5)
containing 0.2 mol/L NaCl. After loading, the column
was washed (flow rate 40.4 mL/h) with 50 mL of the
buffer, followed by washing with 300 mL buffer con-
taining a gradient from 0.2 to 0.5 mol/L NaCl. The
ADE1 activity peak was eluted at 0.32 mol/L NaCl.
The ADE2 pool was applied to a similar column
and washed with a flow rate of 53 mL/h subsequently
with 50 mL Tris-HCl buffer (50 mmol/L, pH 7.5) con-
taining 0.1 mol/L NaCl, and 200 mL Tris-HCl buffer
(50 mmol/L, pH 7.5) containing a gradient from 0.1
mol/L to 0.3 mol/L NaCl. The ADE2 activity peak
eluted at 0.18 mol/L NaCl. The active fractions were
pooled, concentrated with the aid of polyethylene gly-
col (PEG 6000) and dialyzed overnight at 4℃ against
Tris-HCl buffer (50 mmol/L, pH 7.5) and stored at
-30℃ . No activity loss occurred during the dialysis.
1.4 Enzyme and product assays
The activity of allantoate-degrading enzyme was
assayed by modifying the method of Lukaszewski et
al. (1992). The reaction mixture (400 µL) contained
potassium allantoate 10 mmol/L, MnSO4 0.15 mmol/
L, Tricine-NaOH buffer (50 mmol/L, pH 8.5) and an
appropriate amount of enzyme (5-20 µg). Phenylhy-
drazine-HCl (0.0825%, or 5.7 mmol/L) was included
in our assay method (Fig.1, Table 1). The reaction mix-
ture was at 30℃ for assay. The reaction speed done
462 植物生理与分子生物学学报
under the same condition was linear during the assay
period. Controls without enzyme were always included.
The reaction was stopped by the addition of 100 µL
EDTA (10 mmol/L). After the addition of 100 µL so-
dium hydroxide (0.1 mol/L) the mixture was incubated
for 10 min at room tempera ture to hydrolyze
ureidoglycolate to glyoxylate. Then 600 µL potassium
phosphate buffer (0.4 mol/L, pH 7.0) and 200 µL
0.33% (W/V) phenylhydrazine-HCl were added and the
tubes were incubated for 7 min at room temperature.
The tubes were cooled to 0℃ in an ice-water bath.
1 mL HCl (12 mol/L), pre-cooled to –20℃, and 200
µL 1.67% (W/V) potassium ferricyanide solution were
added. After mixing, the tubes were placed at room
temperature for 7 min and the color of the produced
formazan was determined at the wavelength of 535
nm. The molar extinction coefficient of the formed
formazan was 48 050, which was in good agreement
with the value of 49 300 determined by Vogels and
van der Drift (1970). The amount of phenylhydrazine
present in the reaction mixture did not interfere with
the colorimetric determination of ureidoglycolate.
Enzymatic ureidoglycolate degradation was mea-
sured as described by Wells and Lees (1991). Urease
activities in the partially purified ADE2 fractions were
determined at 30℃ in incubation mixtures containing
urea (60 mmol/L), 2-mercaptoethanol (3 mmol/L),
EDTA (2 mmol/L) and 7.5 µg/mL of protein in potas-
sium phosphate buffer (70 mmol/L, pH 7.5). Ammo-
nia released was determined by the phenol-hypochlo-
rite method (Muftic 1964). To determine whether urea
or ammonia was formed as a product of the ALAH
reaction a 5-µL aliquot of the standard ADE reaction
mixture was incubated at 25℃ in a 1- mL mixture con-
taining potassium phosphate buffer (160 mmol/L, pH
7.5), 2-oxoglutarate (5.2 mmol/L), NADH (0.2 mmol/L)
and 25 units of glutamate dehydrogenase (Sigma), both
in the absence or presence of 1.3 units of jack bean
urease (Sigma). Phenylhydrazine-HCl (28.5 µmol/L)
present in the final mixture inhibited neither urease
nor glutamate dehydrogenase. Wells and Lees (1991)
previously showed that much higher phenylhydrazine
concentrations (up to 8 mmol/L) did not influence jack
bean urease activity. One unit (Kat) of enzyme activ-
30 卷
ity is defined as the amount of enzyme that catalyzes
the formation of 1 mol of product per second. Specific
activity is expressed as Kat/mg protein. The protein
content was measured by the method of Bradford
(1976) with bovine serum albumin as the standard.
1.5 Estimation of molecular mass
The molecular mass of ADE2 was estimated by
gel filtration on a Sephacryl S-200 column (1.6 cm ×
97 cm). A mixture of cytochrome c (13.4 kD), soy-
bean trypsin inhibitor (21.5 kD), bovine serum albu-
min (66 kD), ethanol dehydrogenase (141 kD) and
peroxidase (247.5 kD) was used for calibration. The
estimation of ADE2 molecular weight was based on
the data from elution profile of standard protein
markers, from ADE2 elution profile, and from the
mixed elution profile of ADE2 and standard markers.
Cytochrome c showed no ALAH activity.
1.6 Chemical modification
The involvement of certain amino acid residues
in the catalytic action of the enzyme was studied by
testing the effects of chemical modification on its cata-
lytic activity. The residues of lysine, tyrosine, tyrosine
and tryptophan residue(s) and thiol group were modi-
fi e d b y us i ng pyr i dox a l - 5 ’ - pho s pha t e , N -
acetylimidazole, N-bromosuccinamide, Iodoacetic acid
and 5,5’-dithiobis-(2-nitrobenzoic acid). The enzyme
solution (25 µL) was incubated for 10 min at 30℃
with 25 µL of the reagent at a final concentration of
10 mmol/L and then the mixture was used for the
ALAH assay. The amount of modifying agent present
( 1 . 2 5 mmol / L) di d not i nt e r fe r e wi t h t he
ureidoglycolate determination.
2 Results
2.1 Demonstration of allantoate degradation in
crude extracts of French bean seedlings
The inclusion of phenylhydrazine in the incuba-
tion mixture, however, enabled us to demonstrate for
the first time well-defined degradative reaction of
French bean extracts. The presence of phenylhydra-
zine used was crucial: in the absence of phenylhydra-
zine no activity was observed at all (Fig.1); maximal
activity was found between 0.05% and 0.1%. The phe-
nylhydrazine-HCl used in the assay has been optimized
4期 徐志伟等: 四季豆幼苗中尿囊酸酰胺水解酶的一些特性(英文) 463

(Table 1) as phenylhydrazine-HCl also was included fication when the activity of the enzyme fractions ob-
in the ureidoglycolate assay (See section 1.4 Enzyme tained was measured in the presence of 0.15 mmol/L
and product assays). Mn2+ ions (See Partial characterization of the enzyme).
The resulting ADE1 and ADE2 fractions were puri-
fied 274-fold and 579-fold, respectively with yields
5.5% and 11% respectively(Table 2). The ADE1 frac-
t i on s t i l l c ont a i ne d s ome ur e i do gl yc ol a t e
amidohydrolase activity. The ADE2 fraction was free
from ureidoglycolate amidohydrolase, ureidoglycolate
urea-lyase and urease activity.
2.3 ADE2 is allantoate amidohydrolase
To d i s c r i m i n a t e b e t w e e n a l l a n t o a t e
Fig.1 Effect of phenylhydrazine-HCl in reaction mixture on amidohydrolase and allantoicase the formed products
the allantoate-degrading enzyme were quantitized during allantoate degradation by the
Experimental conditions are as given in the section 1.4 En- partially purified ADE2 (Fig.2). It was found that 1
zyme and product assays.
mol of allantoate was degraded to form 1 mol of
ureidoglycolate together with 2 mol of ammonia. The
Table 1 Effect of phenylhydrazine-HCl on the ureidoglycolate stoichiometry did not change when jack bean urease
analysis was added to the incubation mixture. This result con-
Phenylhydrazine-HCl Phenylhydrazine-HCl added Relative firms that ADE2 is an allantoate amidohydrolase,
in reaction mixture (%) in ureidoglycolate assay activity(%) ALAH, since neither ureidoglycolate hydrolyzing en-
0.165 0 28.2 zyme nor urease was present in the partially purified
0.165 100 µL 0.33% 51.7 ADE2.
0.165 200 µL 0.33% 27.8 2.4 Partial properties of the enzyme
0.0825 200 µL 0.33% 100
ADE1 was eluted in the void volume of the
Sephacryl S-200 column and therefore had a molecu-
Extracts prepared from stems and leaves, roots lar mass of over 200 kD. No further attempts were
and cotyledons showed 53%, 30% and 17% of the to- made for a more accurate determination of the mass of
tal allantoate-degrading enzyme activity, respectively. the ADE1 protein. The ADE1 fraction still showed
During germination the specific activity increased from ureidoglycolate hydrolysis activity and most likely
about 6.7 pKat/mg protein in ungerminated seeds to contained aggregated protein, no further research was
about 30 pKat/mg protein in 6-d-old seedlings. attempted. When the ADE1 pool was subjected to chro-
2.2 Isolation of allantoate-degrading enzyme matography on DEAE-Sephadex, the ADE activity was
Similar results were obtained when EDTA-con- found in the fractions with concentration of NaCl be-
taining and EDTA-free buffers were used during puri- tween 0.31 mol/L and 0.34 mol/L with the maximum

Table 2 Purification of allantoate-degrading enzyme from French bean seedlings

Protein Total activity Specific activity Yield Purification
Purification step
(mg) (nKat) (nKat/mg protein) (%) (fold)
Crude extract 1738 40.2 0.023 100 1
(NH4)2SO 4 (25%-50%) 548.8 27.2 0.05 67.8 2.2
Sephacryl S-200, ADE1 122.6 10.7 0.087 26.7 3.8
Sephacryl S-200, ADE2 3.1 7.6 2.45 18.9 106
DEAE-Sephadex A50, ADE1 pool 0.35 2.2 6.29 5.5 274
DEAE-Sephadex A50, ADE2 pool 0.33 4.4 13.33 11 579
464 植物生理与分子生物学学报
Fig.2 Product formation during allantoate degradation
Ureidoglycolate (■) and ammonia in the absence (▲) or pres-
ence (◆) of jack bean urease. Experimental conditions are as
given in the section 1.4 Enzyme and product assays.
activity at 0.32 mol/L, while the ureidoglycolate
amidohydrolase activity was eluted between 0.35 mol/
L and 0.39 mol/L with the maximum at 0.37 mol/L.
The molecular mass of ALAH was estimated to
be 13.5 kD by gel filtration on Sephacryl S-200 (Fig.
3). Maximum activity was observed at pH 8.5 (Fig.4).
The enzyme showed the Michaelis-Menten kinetics
(Fig.5): a Km value for allantoate of 76 µmol/L and a
Vmax of 16.7 nKat/mg protein were found when a par-
tially purified ADE2 pool was used with a specific
activity of 16.4 nKat/mg protein. The product of the
reaction, ureidoglycolate, did not affect the activity in
the ra nge tested (up to 0.2 mmol/L racemic
ureidoglycolate).
The partially purified ALAH did not show any
activity when assayed in the absence of Mn2+ ions.
Addition of Mn2+ ions at concentrations between 0.1
Fig.3 Molecular weight of ALAH (■) was estimated with
standard molecular weight proteins (◆) by gel filtration on a
Sephacryl S-200 column chromatograph
Experimental conditions are as given in the section 1.5 Esti-
mation of molecular mass.
30 卷
Fig.4 Optimum pH in Tricine/NaOH buffer for the enzyme
reaction
Experimental conditions are as given in the section 1.4 Enzyme
and product assays.
Fig.5 Michaelis-Menten kinetics of ALAH
Experimental conditions are as given in the section 1.4 Enzyme
and product assays.
and 0.5 mmol/L resulted in the maximal activity. Ad-
dition of EDTA at a final concentration of 2 mmol/L
to the Mn2+-containing incubation mixture completely
abolished activity. When an aliquot of this reaction
mixture was assayed for enzyme activity (EDTA
0.05 mmol/L; Mn2+ 0.3 mmol/L) full activity was
recovered. Mn2+ ions could be replaced by Cd2+ or Ni2+
(all 0.5 mmol/L), which resulted in 85% and 32% of
the activity measured in the presence of Mn 2+
respectively. However, Mg2+, Ca2+, Cu2+, Co2+ and Fe2+
(all 0.5 mmol/L) could not replace Mn2+ . Cu2+ , Co2+
and Fe2+ (all 0.5 mmol/L) added to the incubation mix-
ture containing 0.15 mmol/L Mn2+ even completely
inhibited the enzyme activity.
After storage for 2 months at -30℃ or 1 week at
4℃ the enzyme lost about 5% and 10% respectively
of its activity. After a heat treatment for 15 min at 55℃
a loss of 15% in activity was observed.
4期 徐志伟等: 四季豆幼苗中尿囊酸酰胺水解酶的一些特性(英文)
However the enzyme was rather resistant to urea.
Pre-treatment with 2 mol/L or 5 mol/L urea for 10 min
at 30℃ only reduced the activity by 5% and 20%,
respectively. A similar treatment with 2 mol/L or 5 mol/
L guanidine resulted in a loss of activity of 10% or
80%, respectively. Pre-treatment with 50 mmol/L SDS
for 10 min at 30℃ resulted in loss of 50% activity.
Strong inhibition to ALAH activity both by
glyoxylate or by glycolate was observed (Fig.6). 50%
inhibition by glyoxlate at about 20 µmol/L or by gly-
colate at about 60 µmol/L.
Fig.6 Effect of glyoxylate (■) and glycolate (▲) on the
enzyme activity
Experimental conditions are as given in the section 1.4 En-
zyme and product assays.
Pyridoxal-5’-phosphate can combine with the ε-
amino group of lysine to form a Schiff base. Pre-incu-
bation of the enzyme with pyridoxal 5’-phosphate (10
mmol/L) for 10 min at 30℃ resulted in the complete
loss of activity (Table 3). N-bromosuccinimide is
group-specific for either tyrosine or tryptophan
residues; Pre-incubation with N-bromosuccinimide
also resulted in total activity loss. N-acetylimidazole
is a specific modifier for tyrosine residue. And pre-
incubation of the partially purified ALAH with
Table 3 Effect of amino acid residual modifier on the allantoate
amidohydrolase
Treatment (10 mmol/L) Residual activity (%)
Control 100
Iodoacetic acid 98.9
’
5,5 -dithiobis-(2-nitrobenzoic acid) 101.5
N-acetylimidazole 94.4
N-bromosuccinimide 0 during ureidoglycolate hydrolysis. A similar explana-
Pyridoxal-5’-phosphate 0
465
N-acetylimidazole had little effect on the enzyme
activity. Iodoacetic acid or 5,5 ’ -dithiobis-(2-
nitrobenzoic acid) are specific modifiers for thiol
group. But pre-incubation with iodoacetic acid or 5,
5’ -dithiobis-(2-nitrobenzoic acid) had no effect on
the activity.
3 Discussion
Ureide catabolism in higher plants has been stud-
ied quite extensively (Thomas and Schrader 1981;
Winkler et al. 1985, 1987). The exact pathway of
allantoate hydrolysis, however, has not been firmly
established and was suggested only on the basis of
physiological studies (Polacco et al. 1982, Winkler et
al. 1987, Stahlhut and Widholm 1989, Stebbins and
Polacco 1995, Vadez and Sinclair 2000). So far no re-
port has appeared to demonstrate allantoate degrada-
tion in crude extracts of French bean. And we failed to
detect allanotoate-degrading enzyme from soybean or
from French bean with those reported non-radiochemi-
cal methods including the method of Lukaszewski et
al (1992).
Addition of phenylhydrazine to the assay mix-
ture was essential for the quantification of allantoate
amidohydrolase activity assay both in crude extract and
in the partially purified ALAH from French bean seed-
lings (Fig.1). It is possible that the method can be used
for me a sur i ng ALAH ( eve n a nd a ll a nt oa t e
amidinohydrolase) from other higher plant. Also
ureidoglycolate amidohydrolase activity in French bean
fruits could be demonstrated only when phenylhydra-
zine was added to the incubation mixtures. For crude
extracts it was assumed that in the absence of phenyl-
hydrazine glyoxylate was converted by enzymes like
glycolate oxidase or aminotransferases, but for pure
enzyme no explanation was given (Wells and Lees
1991).
Both allantoate and ureidoglycolate were not
stable, and they could be non-enzymatic hydrolysized
to form glyoxylate. Since glyoxylate inhibited
allantoate degradation, the most likely function for
phenylhydrazine was to remove glyoxylate formed
tion may be given for the results obtained with
466 植物生理与分子生物学学报
ureidoglycolate amidohydrolase (Wells and Lees
1991).
The mechanism of the strong inhibition both by
glyoxylate or by glycolate (Fig.6) was not investigated
further. For other enzymes inhibited by glyoxylate it
had been shown that either a Schiff base was formed
with a lysine residue resulting in loss of catalytic ac-
tivity (Cook et al. 1985) or that (non)competitive inhi-
bition with substrate occurred (Nimmo 1986). The in-
hibition of pea leaf glycine decarboxylation by
glyoxylate was concentration-dependent but not time-
dependent (Sarojini and Oliver 1987). Either the bind-
ing of the glyoxylate to glycine decarboxylase was
readily reversible or that the formation of the covalent
linkage was very rapid. The first alternative would be
in accordance with the results of the chemical modifi-
cation experiment described above (Table 3). These
result suggested that both lysyl and tryptophanyl resi-
dues were involved in the enzyme catalysis of ALAH.
But neither tyrosine residue nor thiol group is involved
in its enzyme catalysis. Glyoxylate or glycolate might
be bound to an essential lysyl residue to regulate the
allantoate degradation in vivo. Exogenous glyoxylate
(6-10 mmol/L) decreased ureide content in the seed-
lings of ureide-producing lugume Adzuki bean (Xu et
al. 1992). Glyoxylate showed inhibition on allantoin-
ase from the yam and sweet potato (Osuji and Ory
1987). It suggested that there may be some relation-
ships between plant glycolate metabolism and ureide
metabolism.
Partial purification of allantoate-degrading en-
zyme yielded a high- and a low-molecular weight frac-
tion (Table 2). It was indicated that ADE1 and
ureidoglycolate amidohydrolase were present as free
enzymes and not parts of a multifunctional enzyme
complex, differently from the hypothesis in soybeans
by Winkler et al. (1988b). The contaminating
ureidoglycolate amidohydrolase was reported to have
a molecular mass of 300 kD (Wells and Lees 1991). It
was demonstrated that neither ureidoglycolate hydro-
lyzing enzyme nor urease was present in the partially
purified ADE2 fraction (low-molecular fraction). By
product analysis the allantoate-degrading enzyme
present in the low-molecular fraction was identified to
30 卷
be ALAH (Fig.2).
The partially purified ALAH activity required the
presence of Mn2+ ions, similarly to the allantoate-de-
grading activity of the crude extract of soybean
(Winkler et al. 1985).
A high pH optimum and strict dependency on
metal ions for activity was also found for all studied
ALAH’s from bacteria (Vogels and van der Drift 1976,
Xu and van der Drift 1996).
The ALAH was relatively stable in storage. Bac-
terial ALAH’s usually showed a reversible activation/
inactivation behavior (Vogels 1966). Inactivation was
caused by the binding of Mn2+ ions to a non-catalytic
site, whereby binding of these ions to the catalytic site
was prevented. Since the catalytic activity was depen-
dent on Mn2+ ions no enzyme activity was found. The
incorrectly bound ions can be removed either by treat-
ment at pH values below 4 or around pH 6 in the pres-
ence of complexing agents which results in an activa-
tion of the enzyme (van der Drift and Vogels 1969a,
Vogels and van der Drift 1976). Activated enzyme was
inactivated by dilution with EDTA-free buffer (van der
Drift and Vogels 1969b, Xu et al. 1995). So far no
studies on such a behavior were reported for the plant
enzyme. The ALAH from French bean did not show a
reversible activation/inactivation and in this respect
differed from the bacterial enzymes.
Acknowledgements: The authors wish to thank Dr. Chris van
der Drift (University of Nijmegen, Netherlands) and Dr. Peter
Tipton (University of Missouri at Columbia, USA) for their
support in the work on allantoate amidohydrolase.
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