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Original Paper	Pdf file on Synergy omments
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, [email protected]

The photorespiratory enzyme L-serine:glyoxylate amino�transferase (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 chromato�graphy 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 photo����respiration, 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; photo�respiration

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 amino�transferase 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

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 chromato�graphy 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 spectro�metry 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)抯 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 phoshogly�cerate 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抯 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 spectro�metry (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 polyacryl�amide 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抯 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 photo�respiration 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穝)], 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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