Original Paper
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file on Synergy |
Acta Biochim Biophys
Sin 2008, 40: 102-110
doi:10.1111/j.1745-7270.2008.00383.x
Properties of
serine:glyoxylate aminotransferase purified from Arabidopsis thaliana leaves
Maria Kendziorek and Andrzej
Paszkowski*
Department of
Biochemistry, Faculty of Agriculture and Biology, Warsaw University of Life
Sciences-SGGW, Nowoursynowska 159, 02-776 Warsaw, Poland
Received: September
4, 2007
Accepted: October 4,
2007
Abbreviations:
GGAT, L-glutamate:glyoxylate aminotransferase; NCBI, National Center for
Biotechnology Information; PLP, pyridoxal phosphate; SDS-PAGE, sodium dodecyl
sulfate polyacrylamide gel electrophoresis; SGAT, L-serine:glyoxylate
aminotransferase
*Corresponding
author: Tel, 48-22-5932568; Fax, 48-22-5932562; E-mail,
The
photorespiratory enzyme L-serine:glyoxylate aminotransferase (SGAT; EC
2.6.1.45) was purified from Arabidopsis thaliana leaves. The final
enzyme was approximately 80% pure as revealed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis with silver staining. The identity
of the enzyme was confirmed by LC/MS/MS analysis. The molecular mass estimated
by gel filtration chromatography on Sephadex G-150 under non-denaturing
conditions, mass spectrometry (matrix-assisted laser desorption/ionization/time
of flight technique) and sodium dodecyl sulfate-polyacrylamide gel
electrophoresis was 82.4 kDa, 42.0 kDa, and 39.8 kDa, respectively, indicating
dimer as the active form. The optimum pH value was 9.2. The enzyme activity was
inhibited by aminooxyacetate and b-chloro-L-alanine
both compounds reacting with the carbonyl group of pyridoxal phosphate. The
enzyme’s transaminating activity with L-alanine and glyoxylate as
substrates was approximately 55% of that observed with L-serine and
glyoxylate. The lower Km value (1.25
mM) for L-alanine, compared with that of other plant SGATs, and the kcat/Km(Ala) ratio being approximately
2-fold higher than kcat/Km(Ser) suggested that, during photorespiration,
Ala and Ser are used by Arabidopsis SGAT with equal efficiency as amino
group donors for glyoxylate. The equilibrium constant (Keq), derived from the Haldane relation, for the transamination
reaction between L-serine and glyoxylate with the formation of
hydroxypyruvate and glycine was 79.1, strongly favoring glycine synthesis.
However, it was accompanied by a low Km value of
2.83 mM for glycine. A comparison of some kinetic properties of the studied
enzymes with the recombinant Arabidopsis SGATs previously obtained
revealed substantial differences. The ratio of the velocity of the
transamination reaction with L-alanine and glyoxylate as substrates
versus that with L-serine and glyoxylate was 1:1.8 for the native
enzyme, whereas it was 1:7 for the recombinant SGAT. Native SGAT showed a much
lower Km value for L-alanine
compared to the recombinant enzyme.
Keywords L-serine:glyoxylate aminotransferase; glyoxylate
aminotransferase; Arabidopsis thaliana; photorespiration
The hypothesis that two
peroxisomal transaminases, a glutamate:glyoxylate (GGAT; EC 2.6.1.4) and a
serine:glyoxylate aminotransferase (SGAT; EC 2.6.1.45), participate in the
photorespiratory cycle was proposed in 1972 and later confirmed [1,2]. In the
model plant Arabidopsis thaliana, Liepman and Olsen [3] identified two
genes coding for two isoenzymes of GGAT, each with a signal peptide, PTS1, at
the C-terminal, directing them into the peroxisomal compartment. The cDNAs
corresponding to the two GGAT genes were overexpressed in bacteria [3]. The
recombinant GGAT1 and GGAT2 purified from the soluble fraction of
Escherichia coli lysate showed kinetic properties similar to those of
GGAT1 purified from Arabidopsis leaves, and also similar to homologous
enzymes from different plant sources [4–7].
We obtained partially purified GGAT1 and GGAT2 from Arabidopsis leaves,
and studied some of their molecular and kinetic properties [8].
According to Liepman and Olsen, SGAT from Arabidopsis is
encoded by a single gene [9] and they overexpressed in E. coli the cDNA
corresponding to this gene. The kinetic and molecular properties of the
purified protein appeared to be similar to those of homologous plant
aminotransferases [10–15]. To date there has been no information about SGAT purified from
Arabidopsis leaves. Recently, two reports have been published indicating
a crucial role of this enzyme in the photorespiratory cycle and the possibility
of controlling photorespiration by regulation of the expression level of the
gene that encodes SGAT [16,17]. A modulatory effect of light and cytokinin on
the level of SGAT biosynthesis and consequently on the rate of photorespiration
was observed in Spirodela polyrrhiza [16] and Chlamydomonas
reinhardtii [17]. Taler et al [18] identified two genes from Cucumis
melo showing 86% identity of the nucleotide sequence and most probably encoding
two SGAT isoenzymes characterized by 93% amino acid sequence identity.
Transgenic melon plants overexpressing either of these genes showed enhanced L-serine
and L-alanine:glyoxylate aminotransferase activities (both
characteristic for SGAT) and remarkable resistance against an oomycete pathogen
[18]. With at least 44 representatives aminotransferases make up about 1%
of the predicted “metabolism” genes in Arabidopsis [19].
Approximately 60% of the Arabidopsis genes encoding aminotransferases
have been characterized [19]. However, A. thaliana SGAT has not been
purified, and its kinetic and molecular properties have not been studied, which
constitutes a substantial gap in the general knowledge of this model plant
proteome.The present study describes a four-step procedure of purification of
SGAT from Arabidopsis leaves. Molecular and kinetic characteristics of
the enzyme are presented and compared with those of SGATs obtained from other
plant sources [10–15] and also with the recombinant Arabidopsis SGAT purified
from the soluble fraction of E. coli lysate by Liepman and Olsen [9].
The metabolic role of Arabidopsis SGAT is discussed.
Materials and
Methods
Materials
Leaves of A. thaliana (L.) ecotype Landsberg erecta
originating from 31-day-old seed culture grown on a solid medium were used for
study. The 1/2MS medium with the addition of an antibiotic Timentin (0.25 mg/ml; SmithKline
Beecham Pharmaceuticals, Brendford, England) was used [8]. Seeds of A.
thaliana were sterilized in 96% ethanol and 50% sodium hypochlorite, and
after washing they were suspended in 0.1% agarose water solution. The prepared
seeds were placed in Petri dishes (j=11 cm) half-filled with medium (0.5 ml of
seeds per dish) and were kept in a growing chamber (16 h photoperiod, 26/20 ?C,
200 mE). Plants were harvested at the stage of inflorescence shoot
setting.
Purification of SGAT from A. thaliana leaves
All steps except HPLC were carried out at 4 ?C. Finely cut A.
thaliana leaves were homogenized in a type 302 homogenizer (Unipan,
Warsaw, Poland) twice for 30 s each at 7000 rpm in 50 mM K-phosphate buffer, pH
7.5, containing 0.1 mM pyridoxal phosphate (PLP), 0.1 mM phenylmethanesulfonyl
fluoride, 1 mM EDTA, 10 mM 2-mercaptoethanol, and 10% sorbitol (1:5; W/V).
The proteins in the homogenate were fractionated with cooled acetone (–20 ?C). The
fraction precipitated between 40% and 60% acetone concentration was collected
and dissolved in 20 ml of 20 mM K-phosphate buffer, pH 7.5, and centrifuged
at 10,000 g. It was applied to the hydroxyapatite CHT II column (2.6
cm9.0 cm) equilibrated with 20 mM K-phosphate buffer, pH 7.5, and attached to
the Biologic LP chromatography system (Bio-Rad, Hercules, USA). The enzyme was
eluted from the column in 300 ml linear gradient of 20–200 mM K-phosphate buffer,
pH 7.5, at a flow rate of 1 ml/min. To all collected fractions (6 ml), 10 ml of PLP (20 mM)
was added. The fractions showing highest L-serine:glyoxylate activity
were pooled and concentrated using Amicon 8010 (Amicon Inc, Beverly, USA)
supplied with a PM-10 membrane. The concentrated enzyme preparation was
dialyzed against 50 mM Tris/glycine buffer containing 50 mM PLP. The
dialysate was applied to a Protein-Pak Q 8HR anion exchange column (1 cm10 cm)
attached to an HPLC system (Waters, Milford, USA) equilibrated with 50 mM
Tris/glycine buffer, pH 9.1. The enzyme was eluted from the column in 240 ml
linear gradient of 0–0.1 M KCl in column buffer at a flow rate of 1.5 ml/min. To all
collected fractions (4.5 ml), 10 ml of PLP (20 mM) was added. The final
preparation was stored at –80 ?C.
Determination of aminotransferase activities
All aminotransferase activities were calculated from decrease of
NADH absorption at 340 nm measured continuously during the transamination
reaction or after it was terminated. L-aspartate:2-oxoglutarate
aminotransferase activity was determined at 25 ?C in a continuous assay using
NADH and malate dehydrogenase according to Bergmeyer and Bernt [20]. L-alanine:2-oxoglutarate
aminotransferase activity was determined at 25 ?C in a continuous assay using
NADH and lactate dehydrogenase according to Horder and Rej [21]. L-glutamate:glyoxylate
activity was determined at 30 ?C in a discontinuous assay using NADH and
glutamate dehydrogenase according to Rowsell et al [22].For the determination of SGAT activity, transamination was carried
out at 30 ?C in an incubation mixture containing 0.65 ml amino acid (15.4 mM),
5 mM 2-oxoacid, 20 mM PLP, 77 mM K-phosphate buffer, pH 8.0, and the enzymatic protein
(0.2–30.0 mg). The enzyme was pre-incubated with the amino acid substrate for
10 min. The reaction was started by addition of 2-oxoacid, and stopped after 15
min by addition of 0.1 ml of 10% trichloroacetic acid. The rate of the reaction
using glycine as the amino group donor was estimated by determination of the
remaining 2-oxoacid substrates after transamination was stopped so it required
lowering the initial concentration of hydroxypyruvate and pyruvate to 0.5 mM.
2-Oxoacids (products or the remaining substrates) were determined by the
spectrophotometric method using NADH and lactate dehydrogenase according to
Rowsell et al [22].SGAT specific activity (1 U/mg) was expressed as 1 mmol of the
oxoacid product formed per minute and per mg protein at 30 ?C. Protein was
determined according to Bradford with bovine serum albumin as a standard [23].
Determination of molecular mass
Determination of molecular mass
The molecular mass of SGAT under native conditions was estimated on
a Sephadex G-150 column (2.6 cm 81 cm) equilibrated with 50 mM Tris/glycine
buffer, pH 9.1, using the enzyme preparation obtained after the acetone
fractionation step of the purification procedure. The fractions (3 ml) were
collected at a rate of 12 ml/h. The column was calibrated with blue dextran
2000 (2000 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin dimer
(134 kDa) and monomer (67 kDa), ovalbumin (43 kDa), and chymotrypsinogen (25
kDa). The molecular mass of purified SGAT was determined by mass spectrometry
using the matrix-assisted laser desorption/ionization/time of flight technique
with a Reflex IV mass spectrometer (Department of Neurobiochemistry, Faculty of
Chemistry, Jagiellonian University, Krak?w, Poland).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
The gels were prepared and run according to Laemmli [24]. Protein
bands were silver stained according to the method of Blum et al [25].
The gels were calibrated with Bio-Rad low molecular mass standards (14.4–94.7 kDa). The
gels with stained protein bands were scanned and analyzed by a computer
software package, NIH Image 1.62 for Macintosh (available at http://rsb.info.nih.gov/nih-image/).
Native PAGE and detection of
enzymatic activity in gels
Polyacrylamide gel (7.5%) was prepared in 50 mM Tris/glycine buffer,
pH 9.1, containing 10% glycerol. Electrophoresis was run in the same buffer
without glycerol. Gels were stained for the serine:glyoxylate aminotransferase
activity as described previously [15] using NADH and lactate dehydrogenase.
Identification of purified SGAT
The silver-stained proteins were eluted separately from the SDS gel,
digested by trypsin and analyzed by liquid chromatography coupled to a
quadrupole time-of-flight mass spectrometer in the Laboratory of Mass
Spectrometry (Institute of Biochemistry and Biophysics, Polish Academy of
Sciences, Warsaw, Poland). The results were used to search SwissProt (http://www.expasy.org/sprot) and the
MSDB protein sequence database (http://www.matrixscience.com/help/esq_db_setup_msdb.html)
using the MASCOT program (http://www.matrixscience.com/search_from_select.html).
Results
Purification of Arabidopsis
SGAT
The procedure used to purify SGAT from A. thaliana leaves is
presented in Table 1. Inclusion of an acetone precipitation step
resulted in substantial (almost 4-fold) increase in specific activity (Table
1), similar to results obtained with maize and wheat SGAT [15]. It should
be noted that treatment with acetone completely destroyed the activities of
three aminotransferases: glutamate:glyoxylate (0.306 U/mg protein);
alanine:2-oxoglutarate (0.435 U/mg protein); and aspartate:2-oxoglutarate
(0.967 U/mg protein) present in the initial homogenate. An approximately 6-fold
increase in specific activity was achieved using a hydroxyapatite column (Table
1). The anion exchange HPLC Protein-Pak Q 8HR column, used in an earlier
report during the purification of maize SGAT [15], confirmed its high
efficiency (Table 1). It should be noted that the purification did not
yield any suggestion of other isoforms of the Arabidopsis enzyme. The
specific L-serine:glyoxylate activity of the final enzyme preparation
exceeded 64-fold of that of the initial leaf homogenate (Table 1).
Homogeneity and molecular mass
determination
SDS-PAGE of the final enzyme preparation with silver staining
showed a main protein band of a molecular mass of 39.80.9 kDa, estimated by
densitometric determination (data not shown) to constitute approximately 79.2%
of the total amount of loaded protein (3 mg), and three additional
bands (20.8%) of lower molecular masses (Fig. 1). The native molecular
mass of the studied enzyme was estimated to be 82.41.3 kDa by gel filtration chromatography
on a Sephadex G-150 column under non-denaturing conditions (Fig. 2).
The results from SDS-PAGE and the Sephadex G-150 column were verified by mass
spectrometry using the matrix-assisted laser desorption/ionization/time of
flight technique. The molecular mass of the SGAT subunit estimated by this
method was 42.012 kDa.
Identification of SGAT by LC/MS/MS analysis
The identity of the main band and its closest band (15.2% of the
total amount of protein) of a slightly lower molecular mass (Fig. 1) was
determined by mass spectrometry. Each of these two protein bands was separately
eluted from the gel and subjected to trypsin digestion. The resulting peptide
mixture was analyzed using LC/MS/MS. The identification score for SGAT was 279.
A comparison of the obtained results with the National Center for Biotechnology
Information (NCBI)? A. thaliana protein sequence database (http://www.ncbi.nlm.nih.gov/)
showed that four of all the peptides obtained from the main band showed 100%
identity with fragments of SGAT amino acid sequence (NCBI protein accession No.
BAB20811). These peptides represented 12.5% of the whole amino acid sequence of
the enzyme from Arabidopsis. The trypsin hydrolysate of the minor band
did not contain any peptides similar to the Arabidopsis SGAT amino acid
sequence, as indicated by mascot.
However, in the hydrolized sample (minor band), we found 14 peptides which were
identified by MASCOT as fragments of putative phoshoglycerate kinase (NCBI
protein accession No. AAM61185) (data not shown).
Electrophoretic mobility
The electrophoretic mobility of the studied enzyme at pH 9.1 was
compared with that of the aminotransferases purified by us earlier from maize
and wheat leaves [15].A zymogram of the SGAT activity developed after native PAGE of the
partially purified SGATs from maize, wheat, and A. thaliana leaves is
presented in Fig. 3. The SGAT from Arabidopsis occupied a
position between the maize and wheat enzymes (Fig. 3).
Kinetic studies
The activity of purified SGAT was tested with different pairs of
substrates: L-amino acid:2-oxoacid (Table 2). The highest
reaction rate was achieved with 15.4 mM L-serine and 5 mM glyoxylate as
substrates; approximately half rate was observed when L-alanine and
glyoxylate were used at the same concentrations (Table 2). The lower
activities (approximately 10% of that with 15.4 mM L-serine and 0.5 mM
glyoxylate) were seen with 15.4 mM glycine and hydroxypyruvate or pyruvate as
substrates, both with 2-oxoacids at 0.5 mM concentration (Table 2). SGAT
from Arabidopsis, similar to the enzyme from wheat and unlike SGATs from
maize [15] and Hyphomicrobium methylovorum [26], was not able to
catalyze the reaction using ketomalonate as the amino group acceptor (Table
2).The optimum pH value for the Arabidopsis SGAT was 9.2 (Fig.
4), the highest value ever determined for a plant glyoxylate
aminotransferase.Two inhibitors reacting with the carbonyl group of pyridoxal
phosphate, the prosthetic group of aminotransferases, aminooxyacetate and
-chloro-L-alanine, inhibited the enzyme, aminooxyacetate being much more
effective (Table 3). The other inhibitor, p-hydroxymercuribenzoate,
which reacts with the sulfhydryl groups, did not inhibit SGAT activity
significantly at 0.1 mM concentration, consistent with the specificity of its
inhibitory action (Table 3). The 24.1% decrease in enzyme activity
achieved using 1.0 mM inhibitor might be considered to be unspecific (Table
3). The turnover parameter (kcat) and the
ratio of kcat/Km were
calculated for Arabidopsis SGAT. To obtain the molar concentration, the
enzyme?
molecular mass estimated by SDS-PAGE (39.8 kDa) (Fig. 1), as well as the
results of a densitometric analysis of the gel after SDS-PAGE, showing approximately
20.8% of unrelated protein contamination, was taken into account. The Vmax values were calculated using the Michaelis-Menten equation. The
values of the apparent constants Km
(further called Km) were determined for the substrates
of forward and reverse reactions: L-serine or L-alanine;
glyoxylate and glycine; and hydroxypyruvate or pyruvate, respectively, using
the Lineweaver-Burk plot (Table 4).The experiment was carried out using 0.5 mM or 10 mM glyoxylate with
four L-serine concentrations (0.5–7.5 mM) and 30 mM L-serine
with four glyoxylate concentrations (0.1–7.0 mM), or 0.5 mM
hydroxypyruvate with four glycine concentrations (0.5–7.5 mM) and 15.4 mM glycine
with four hydroxypyruvate concentrations (0.15–0.30 mM). Furthermore, Km values for L-alanine and glyoxylate were estimated with the
use of 0.5 mM or 10 mM glyoxylate with five L-alanine concentrations
(0.1–7.5 mM) and 30 mM L-alanine with four glyoxylate
concentrations (0.1–3.5 mM) or 0.5 mM pyruvate with four glycine concentrations (0.5–15.4 mM) and
15.4 mM glycine with four pyruvate concentrations (0.1–0.5 mM) (Table 4).
The unprecedented low values for Km(Gly)
and Km(Ala) are noteworthy (Table 4). The kcat and kcat/Km values for the Arabidopsis SGAT (Table 4) were
distinctly lower than those from the maize and wheat SGATs [15]. In addition,
it should be noted that the kcat/Km(Ala) value was almost 2-fold higher than the corresponding value for
serine (Table 4).The equilibrium constant (Keq) for
the transamination reaction between L-serine and glyoxylate with the
formation of hydroxypyruvate and glycine (initial concentrations of substrates:
amino acids, 15.4 mM; 2-oxoacids, 0.5 mM), calculated from the Haldane equation
[27].
Eq.
It was equal to 79.1, strongly favoring glycine formation. Keq calculated for the reaction between L-alanine and glyoxylate
with the formation of pyruvate and glycine (substrates at the same initial
concentrations as above) was equal to 21.8.
Discussion
To our knowledge, our report is the first to describe a simple,
four-step procedure of purification of SGAT from the leaves of model plant A.
thaliana. The inclusion of the acetone step into the purification scheme
resulted in a good yield (63.4%), similar to results found in maize and wheat
[15]. The extraordinary stability of the maize, wheat and Arabidopsis
SGATs in 60% acetone, in contrast to the aspartate (EC 2.6.1.1), alanine (EC 2.6.1.2)
and glutamate:glyoxylate (GGAT; EC 2.6.1.4) aminotransferases from maize, wheat
[15], and A. thaliana leaves, suggests high hydrophobicity of their
surfaces. The presence of hydrophobic groups on the surface of a protein
molecule is known to protect from the denaturing action of acetone [28]. We
aligned the Arabidopsis SGAT amino acid sequence (NCBI protein accession
No. BAB20811) deduced from the cDNA sequence and those of the maize and wheat
SGATs reconstructed on the basis of the expressed sequence tags from the TIGR
database (http://compbio.dfci.harvard.edu/tgi/).
The maize and wheat enzymes showed 81% and 84% identity with SGAT from A.
thaliana, respectively (using the ClustalW algorithm available at http://www.ebi.ac.uk/Tools/clustalw2/index.html).
The mean hydropathy indexes of the maize, wheat, and Arabidopsis SGAT
amino acid sequences calculated according to the method of Kyte and Doolittle
were 0.024, 0.020, and 0.023, respectively, each distinctly above the average
value (–0.4) reported by those authors for 84 fully sequenced soluble
enzymes [29]. This result confirmed the high hydrophobicity of these three
aminotransferases [30] and explained their stability in acetone.A comparison of the molecular mass of Arabidopsis SGAT
determined under denaturing conditions (39.8 kDa), the results of molecular
filtration of the active enzyme (82.4 kDa), the molecular mass obtained from
mass spectrometry (42.0 kDa), and the reported molecular mass of SGATs from
other plants
[5,11,12,15] indicates that these aminotransferases are
all dimers. We have no straightforward explanation for the discrepancy between
the results of our SDS-PAGE molecular mass estimation and the average
molecular mass determined under denaturing conditions for several SGATs from
different plant sources (approximately 45 kDa) [12,15] or the results of
theoretical calculation on the basis of the Arabidopsis SGAT amino acid
sequence (about 44 kDa). At least part of the explanation could lie in the
aliphatic index (relative volume of a protein occupied by aliphatic side
chains) of the Arabidopsis aminotransferase, calculated according to
Ikai [31], which exceeds those for maize and wheat SGATs by more than 3%.
It has been shown that increasing the number of aliphatic amino acids (even by
only one residue) increases the mobility of polypeptides on SDS polyacrylamide
gels [32]. It is difficult to take into consideration the results of SDS-PAGE
molecular mass determination of the recombinant Arabidopsis SGAT shown
by Liepman and Olsen because it seems that their data were obtained by
calibration against only two markers [9].It should be noted that the values of the theoretical isoelectric
point calculated on the basis of the amino acid sequences [33] of maize, wheat
and Arabidopsis SGATs are 6.72, 8.44, and 7.69, respectively, in
agreement with their behaviour during native PAGE at pH 9.1.
Our inhibitor studies showed that Arabidopsis SGAT requires
PLP for its catalytic activity, as other plant glyoxylate aminotransferases do
[6,11,13,15]. Results also suggested the probable absence of any sufhydryl
groups at the active site of the enzyme, similar to SGATs from various other
plant sources [10,13–15]. The studied enzyme showed an extraordinary high optimum pH
level of 9.2. We have recently proposed that this might, to some extent, be a
consequence of the studied plant? evolution under specific climatic conditions [15].The ratio of the velocity of the transamination reaction with L-alanine
and glyoxylate as substrates versus that with L-serine and glyoxylate
was 1:1.8 for the native enzyme studied here compared with 1:7 for the
recombinant Arabidopsis SGAT obtained by Liepman and Olsen [9]. Native
SGAT showed the lowest Km values for L-alanine
(1.25 mM) and glycine (2.83 mM) among those determined for plant SGATs up to
now [7,10,13,15]. Its affinity for L-alanine was 80-fold higher than
that of the recombinant enzyme [9]. All of these differences indicate that
some stages of the maturation of the recombinant SGAT might be disturbed. It is
known that bacteria lack the enzyme systems to carry out the specific
post-translational processing that many eukaryotic proteins require for
biological activity [34].At very low substrate concentrations, the ratio of kcat/Km behaves as a second order rate constant for
the reaction between the substrate and free enzyme. It provides a direct
measure of the enzyme’s efficiency and specificity [35]. A comparison of the
calculated values of kcat/Km for Arabidopsis SGAT with two different amino acid
substrates, L-alanine or L-serine, unexpectedly indicates L-alanine
as the preferred substrate for the studied enzyme under partial saturation conditions.
Ohkama-Ohtsu et al reported that the physiological concentration of L-serine
in A. thaliana leaves is only twice that of L-alanine [36].
Considering the fact that kcat/Km(Ala) was almost 2-fold higher than kcat/Km(Ser), one can speculate that during photorespiration in Arabidopsis,
L-alanine and L-serine are used by SGAT with equal efficiency.
Several authors have proposed L-alanine as a direct and important amino
group donor for photorespiratory glycine formation, however, they all suggested
preferential use of this amino acid by GGAT, another photorespiratory
glyoxylate transaminase [3,4,37,38]. The range of the two calculated kcat/Km values is far below the diffusion-controlled
limit [108 to 109 1/(M?)], indicating the applicability of the Michaelis-Menten equilibrium
assumption for the studied reactions [35]. It should also be noted that the kcat/Km values for L-alanine and L-serine
in transamination with glyoxylate are substantially lower than those found for
SGATs from maize and wheat catalyzing the amino group transfer between L-serine
and glyoxylate [15]. It is likely that the low kcat/Km values for the Arabidopsis SGAT are related to the
relatively low intensity of the photorespiration in the A. thaliana
leaves when compared with that in maize and wheat seedlings.We observed low reaction rates with glycine and hydroxypyruvate or
pyruvate as substrates. The Keq derived from the
Haldane relation [27] for the transamination between L-serine and
glyoxylate catalyzed by purified Arabidopsis SGAT was 79.1. When L-alanine
was used as the amino group donor, Keq
equaled 21.8. This is not surprising because glyoxylate has long been known to
react readily, non-enzymatically, with pyridoxamine [39]. The high values of Keq indicate that in physiological conditions the reverse reactions
with glycine as the amino acid substrate proceeds with a positive free energy
change. However, it does not exclude some physiological importance of these
reactions that might function in the dark when glyoxylate concentration is low.
The extraordinarily low Km value for glycine for Arabidopsis
SGAT, when compared with the results obtained from other plant SGATs [4,10,13],
supports this hypothesis. Thornton observed a greater content of labeled N in
Ser than in all amino acids, other than Gly and Ser, in the root amino acid
pool of Lolium perenne following a 3 h uptake of Gly [40]. It is
suggested that SGAT must have been used to synthesize Ser to some degree [40].In summary, the consistency of our observations on the Arabidopsis
SGAT hydrophobicity and electrophoretic mobility, with the results of
theoretical calculations carried out on the basis of the amino acid sequence,
is noteworthy. The comparison of the substrate specificity and affinity for L-alanine
of the native enzyme studied here and the recombinant one obtained by Liepman
and Olsen [9] indicates that during SGAT production in bacteria some steps of
its maturation required for full catalytic activity could be disturbed.
Furthermore, the low value of Km(Ala) of 1.25 mM,
accompanied by kcat/Km(Ala) almost 2-fold higher than kcat/Km(Ser), indicates that Arabidopsis SGAT is able to use L-alanine
along with L-serine as an amino group donor for glyoxylate during photorespiration.
Moreover, the low Km(Gly) of 2.83 mM suggests that
the reactions in the direction of L-serine or L-alanine
production might be of physiological importance in spite of the high
equilibrium constants for the reverse transamination.
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