Glutamate and serine as competing donors for amination of glyoxylate in leaf peroxisomes

When provided with glycollate, peroxisomal extracts of leaves of spinach beet (Beta vulgaris L. cv.) converted L-serine and L-glutamate to hydroxypyruvate and 2-oxoglutarate respectively. When approximately saturating concentrations of each of these amino acids were incubated separately with glycollate, the utilization of serine was greater than that of glutamate. The utilization of glutamate was substantially reduced by the presence of relatively low concentrations of serine in the reaction mixture, whereas even high concentrations of glutamate caused only small reductions in serine utilization. Over the entire range of concentrations of amino acids examined, serine was invariably the preferred amino-group donor, but this preference was abolished at higher concentrations of glyoxylate. Serine not only competed favourably for glyoxylate but also inhibited L-glutamate: glyoxylate aminotransferase (GGAT), the degree of inhibition depending upon the glyoxylate concentration. Studies of L-serine: glyoxylate aminotransferase (SGAT) and GGAT in partially purified extracts from spinach-beet leaves confirmed that serine competitively inhibited GGAT but glutamate did not affect SGAT. Both enzymes were inhibited by high glyoxylate concentrations, the inhibition being relieved by suitably high concentrations of the appropriate amino acid. It is concluded that at the low glyoxylate concentrations likely to occur in vivo, the preferential utilization of serine would ensure flux through the glycollate pathway to glycerate, but at higher concentrations of glyoxylate, both enzymes could be fully active in glyoxylate amination.Abbreviations SGAT L-serine: glyoxylate aminotransferase - GGAT L-glutamate: glyoxylate aminotransferase     

Glutamate:glyoxylate aminotransferase modulates amino acid content during photorespiration

In photorespiration, peroxisomal glutamate:glyoxylate aminotransferase (GGAT) catalyzes the reaction of glutamate and glyoxylate to produce 2-oxoglutarate and glycine. Previous studies demonstrated that alanine aminotransferase-like protein functions as a photorespiratory GGAT. Photorespiratory transamination to glyoxylate, which is mediated by GGAT and serine glyoxylate aminotransferase (SGAT), is believed to play an important role in the biosynthesis and metabolism of major amino acids. To better understand its role in the regulation of amino acid levels, we produced 42 GGAT1 overexpression lines that express different levels of GGAT1 mRNA. The levels of free serine, glycine, and citrulline increased markedly in GGAT1 overexpression lines compared with levels in the wild type, and levels of these amino acids were strongly correlated with levels of GGAT1 mRNA and GGAT activity in the leaves. This accumulation began soon after exposure to light and was repressed under high levels of CO(2). Light and nutrient conditions both affected the amino acid profiles; supplementation with NH(4)NO(3) increased the levels of some amino acids compared with the controls. The results suggest that the photorespiratory aminotransferase reactions catalyzed by GGAT and SGAT are both important regulators of amino acid content.     

Properties of serine：glyoxylate aminotransferase purified from Arabidopsis thaliana leaves

The photorespiratory enzyme L-serine：glyoxylate amino- transferase （SGAT; EC 2.6.1.45） was purified from Arabidopsis thaliana leaves. The f＇mal enzyme was approximately 80 % pure as revealed by sodium dodecyl sulfatepolyacrylamide 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 β-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 Kmvalue （1.25 mM） for L-alanine, compared with that of other plant SGATs, and the kcat/Km（Ala） ratio being approxi- mately 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.     

Serine:glyoxylate aminotransferase from the seedlings of rye (Secale cereale L.)

Serine:glyoxylate aminotransferase (EC 2.6.1.45) from green parts of 7-day-old rye seedlings was purified 600-fold. Specific activity of the purified enzyme against L-serine and glyoxylate as substrates was 53.2 mumol/mg protein per minute at 30 degrees C. The enzyme activity with L-alanine or L-asparagine and glyoxylate, or with L-asparagine and hydroxypyruvate was 20% that with L-serine and glyoxylate as the amino group acceptor, whereas with L-alanine or glycine and hydroxypyruvate it was 10% of that value. The reaction rate with pyruvate and L-asparagine, glycine or L-serine was very low. The enzyme was stabilized by the presence of sucrose, pyridoxal phosphate and 2-mercaptoethanol. Molecular sieving of the native enzyme on Sephacryl S-300 gel gave Mr values of 91,200 and 85,000, whereas the molecular weight estimated by SDS-polyacrylamide gel electrophoresis was 43,000, indicating the dimeric structure of the enzyme.     

Glutamate:glyoxylate aminotransferase from the seedlings of rye (Secale cereale L.)

Glutamate:glyoxylate aminotransferase from green parts of 7-day-old rye seedlings was purified almost to homogeneity. Specific activity of the purified enzyme measured with L-glutamate and glyoxylate as substrates, was 46.1 units/mg. The enzyme activity with L-alanine and 2-oxoglutarate as substrates was higher by a factor of 1.5, whereas with L-alanine and glyoxylate or L-glutamate and pyruvate it was similar to that with L-glutamate and glyoxylate. L-Aspartate, L-arginine and L-ornithine could also serve as substrate. The reaction followed the Ping-Pong Bi Bi mechanism and Km values for L-glutamate and glyoxylate were 2.6 and 0.5 mM, respectively. Pyridoxal phosphate was found to be the coenzyme of glutamate-glyoxylate aminotransferase. This coenzyme was rather tightly bound with the enzyme protein, as the attempts at its complete resolution from the apoenzyme were unsuccessful. Pyridoxal phosphate, 2-mercaptoethanol and sucrose, or bovine serum albumin stabilized the enzyme. Molecular weight of glutamate:glyoxylate aminotransferase from rye seedlings, determined by SDS-polyacrylamide gel electrophoresis, was 58,800 +/- 2,100, whereas molecular sieving on Sephacryl S-200 gel gave values of 70,800 +/- 700 or 61,400. Similar values obtained for the denatured and nondenatured enzyme seem to indicate that it is a monomeric protein.     

光呼吸途径中两种转氨酶的研究进展

植物丝氨酸：乙醛酸氨基转移酶（SGAT）与谷氨酸：乙醛酸氨基转移酶（GGAT）主要催乙醛酸的转氨反应,是光呼吸途径中的两种关键酶。此两种酶大都为二聚体,在高等植物体内主要位于过氧化物酶体内,而在真核藻类植物体内则位于线粒体内,对植物的生长发育与抗逆性具有重要影响。本文对SGAT与GGAT在植物光合作用、氨基酸代谢和抗逆性等方面的研究进展进行了综述,以期对SGAT与GGAT的研究有所帮助。     

Serine:Glyoxylate Aminotransferases from Maize and Wheat Leaves: Purification and Properties

Photorespiratory enzyme serine:glyoxylate aminotransferase (SGAT, EC 2.6.1.45) was purified from green parts of seedlings of two Gramminae species with different photosynthetic pathways, maize (Zea mays L., C(4) species) and wheat (Triticum aestivum L., C(3) species). The preparation from wheat was homogeneous as judged by SDS-PAGE with silver staining for proteins; however, the same method revealed approximately 9% contamination in a highly purified maize preparation. Molecular masses of SGAT from maize and wheat were estimated by SDS-PAGE to be 44.1 and 44.6 kDa, respectively. C(4) enzyme exhibited a specific activity in homogenates that was seven times lower than wheat, and this was associated with lower K (m) values for all substrates examined as well as a more than two times lower turnover number k (cat) with serine and glyoxylate as a pair of substrates. In contrast, the ratio of the turnover number to K (m)(Ser)(k (cat)/K (m)(Ser)) for C(4) aminotransferase proved to be about two times higher than for C(3) aminotransferase. The sensitivity of two enzymes to some inhibitors, especially aminooxyacetate, was different and they also differed with respect to thermal stability and pH optimum - the maize enzyme required 0.6 unit higher pH (8.6) for maximal activity and was more heat-resistant.     

Biochemical characterization and kinetic properties of alanine aminotransferase homologues partially purified from wheat (Triticum aestivum L.)

Four homologues of alanine aminotransferase have been isolated from shoots of wheat seedlings and purified by saline precipitation, gel filtration, preparative electrophoresis and anion exchange chromatography on Protein-Pak Q 8HR column attached to HPLC. Alanine aminotransferase 1 (AlaAT1) and 2 (AlaAT2) were purified 303- and 452-fold, respectively, whereas l-glutamate: glyoxylate aminotransferase 1 (GGAT1) and 2 (GGAT2) were purified 485- and 440-fold, respectively. Consistent inhibition of AlaAT (EC 2.6.1.2) and GGAT (EC 2.6.1.4) activities by p-hydroxymercuribenzoate points on participation of cysteine residues in the enzyme activity. The molecular weight of AlaAT1 and AlaAT2 was estimated to be 65 kDa and both of them are monomers in native state. Nonsignificant differences between K_m using alanine as substrate and catalytic efficiency (k_cat/K_m) for l-alanine in reaction with 2-oxoglutarate indicate comparable kinetic constants for AlaAT1 and AlaAT2. Similar kinetic constants for l-alanine in reaction with 2-oxoglutarate and for l-glutamate in reaction with pyruvate for all four homologues suggest equally efficient reaction in both forward and reverse directions. GGAT1 and GGAT2 were able to catalyze transamination between l-glutamate and glyoxylate, l-alanine and glyoxylate and reverse reactions between glycine and 2-oxoglutarate or pyruvate. Both GGATs also consisted of a single subunit with molecular weight of about 50 kDa. The estimated K_m for GGAT1 (3.22 M) and GGAT2 (1.27 M) using l-glutamate as substrate was lower in transamination with glyoxylate than with pyruvate (9.52 and 9.09 mM, respectively). Moreover, distinctively higher values of catalytic efficiency for l-glutamate in reaction with glyoxylate than for l-glutamate in reaction with pyruvate confirm involvement of these homologues into photorespiratory metabolism.     

Identification of photorespiratory glutamate:glyoxylate aminotransferase (GGAT) gene in Arabidopsis

In the photorespiratory process, peroxisomal glutamate:glyoxylate aminotransferase (GGAT) catalyzes the reaction of glutamate and glyoxylate to 2-oxoglutarate and glycine. Although GGAT has been assumed to play important roles for the transamination in photorespiratory carbon cycles, the gene encoding GGAT has not been identified. Here, we report that an alanine:2-oxoglutarate aminotransferase (AOAT)-like protein functions as GGAT in peroxisomes. Arabidopsis has four genes encoding AOAT-like proteins and two of them (namely AOAT1 and AOAT2) contain peroxisomal targeting signal 1 (PTS1). The expression analysis of mRNA encoding AOATs and EST information suggested that AOAT1 was the major protein in green leaves. When AOAT1 fused to green fluorescent protein (GFP) was expressed in BY-2 cells, it was found to be localized to peroxisomes depending on PTS1. By screening of Arabidopsis T-DNA insertion lines, an AOAT1 knockout line (aoat1-1) was isolated. The activity of GGAT and alanine:glyoxylate aminotransferase (AGAT) in the above-ground tissues of aoat1-1 was reduced drastically and, AOAT and glutamate:pyruvate aminotransferase (GPAT) activity also decreased. Peroxisomal GGAT was detected in the wild type but not in aoat1-1. The growth rate was repressed in aoat1-1 grown under high irradiation or without sugar, though differences were slight in aoat1-1 grown under low irradiation, high-CO2 (0.3%) or high-sugar (3% sucrose) conditions. These phenotypes resembled those of photorespiration-deficient mutants. Glutamate levels increased and serine levels decreased in aoat1-1 grown in normal air conditions. Based on these results, it was concluded that AOAT1 is targeted to peroxisomes, functions as a photorespiratory GGAT, plays a markedly important role for plant growth and the metabolism of amino acids.     

Short communication. Serine: glyoxylate aminotransferase exerts no control on photosynthesis

A barley (Hordeum vulgare L.) mutant deficient in serine:glyoxylate aminotransferase (SGAT) was crossed with wild-type plants to generate heterozygous mutants. Plants of the F2 generation with reduced SGAT activities (45-60% of wild-type activities) contained proportionally less SGAT protein. Reduced SGAT activities resulted in the accumulation of serine and, to a smaller extent, of glycine, indicating that the flux through the photorespiratory pathway was restricted. Rates of photosynthesis were, however, not affected by the reduction in SGAT activity.     

Characteristics of alanine: glyoxylate aminotransferase from Saccharomyces cerevisiae, a regulatory enzyme in the glyoxylate pathway of glycine and serine biosynthesis from tricarboxylic acid-cycle intermediates.

Alanine: glyoxylate aminotransferase (EC 2.6.1.44), which is involved in the glyoxylate pathway of glycine and serine biosynthesis from tricarboxylic acid-cycle intermediates in Saccharomyces cerevisiae, was highly purified and characterized. The enzyme had Mr about 80 000, with two identical subunits. It was highly specific for L-alanine and glyoxylate and contained pyridoxal 5'-phosphate as cofactor. The apparent Km values were 2.1 mM and 0.7 mM for L-alanine and glyoxylate respectively. The activity was low (10 nmol/min per mg of protein) with glucose as sole carbon source, but was remarkably high with ethanol or acetate as carbon source (930 and 430 nmol/min per mg respectively). The transamination of glyoxylate is mainly catalysed by this enzyme in ethanol-grown cells. When glucose-grown cells were incubated in medium containing ethanol as sole carbon source, the activity markedly increased, and the increase was completely blocked by cycloheximide, suggesting that the enzyme is synthesized de novo during the incubation period. Similarity in the amino acid composition was observed, but immunological cross-reactivity was not observed among alanine: glyoxylate aminotransferases from yeast and vertebrate liver.     

Photorespiratory N donors,aminotransferase specificity and photosynthesis in a mutant of barley deficient in serine: glyoxylate aminotransferase activity

A mutant of Hordeum vulgare L. (LaPr 85/84) deficient in serine: glyoxylate aminotransferase (EC 2.6.1.45) activity has been isolated. The plant also lacks serine: pyruvate aminotransferase and asparagine: glyoxylate aminotransferase activities. Genetic analysis of the mutation strongly indicates that these three activities are all carried on the same enzyme protein. The mutant is incapable of normal rates of photosynthesis in air but can be maintained at 0.7% CO₂. The rate of photosynthesis cannot be restored by supplying hydroxypyruvate, glycerate, glutamate or ammonium sulphate through the xylem stream. This photorespiratory mutant demonstrates convincingly that photorespiration still occurs under conditions in which photosynthesis becomes insensitive to oxygen levels. Two major peaks and one minor peak of serine: glyoxylate aminotransferase activity can be separated in extracts of leaves of wild-type barley by diethylaminoethyl-sephacel chromatography. All three peaks are missing from the mutant, LaPr 85/84. The mutant showed the expected rate (50%) of ammonia release during photorespiration but produced CO₂ at twice the wild-type rate when it was fed [¹⁴C]glyoxylate. The large accumulation of serine detected in the mutant under photorespiratory conditions shows the importance of the enzyme activity in vivo. The effect of the mutation on transient changes in chlorophyll a fluorescence initiated by changing the atmospheric CO₂ concentration are presented and the role of the enzyme activity under nonphotorespiratory conditions is discussed.Abbreviations DEAE diethylaminoethyl - PFR photon fluence rate - SGAT serine:glyoxylate aminotransferase     

Development of glutamate:glyoxylate aminotransferase in the cotyledons of cucumber (Cucumis sativus) seedlings.

Glutamate:glyoxylate aminotransferase had been reported to be present exclusively in the peroxisomes of plant leaves and to participate in the glycollate pathway in leaf photorespiration (Tolbert (1971) Annu. Rev. Plant Physiol. 22, 45-74]. Glutamate:glyoxylate aminotransferase activity was already present in the etiolated cotyledons of cucumber (Cucumis sativus) seedlings, and increased during greening. The enzyme was present only in the cytosol of the etiolated cotyledons and appeared in the peroxisomes during greening. The enzyme was purified to homogeneity from the cytosol of the etiolated cotyledons and from the peroxisomes of the green cotyledons of cucumber seedlings. The two enzyme preparations had nearly identical enzymic and physical properties. On the basis of these findings, roles of glutamate:glyoxylate aminotransferase in the glycollate pathway in photorespiration, and the mechanism of its appearance in the peroxisomes during greening, are discussed.     

Subcellular distribution, multiple forms and development of glutamate-pyruvate (glyoxylate) aminotransferase in plant tissues

According to a sucrose density gradient analysis of cell organelles from homogenates of green leaves of rye, wheat and pea seedlings glutamate-pyruvate aminotransferase was predominantly localized in the leaf microbodies (peroxisomes; 90%) and to a minor extent in the mitochondria (10%) but completely absent from chloroplasts. In etiolated rye leaves the distribution of the enzyme was similar. In other non-green tissues glutamate-pyruvate aminotransferase was predominantly associated with the mitochondria but also present in the microbodies of dark-grown pea roots and in the glyoxysomes of Ricinus endosperm. In the microbodies isolated from potato tubers the enzyme was not detectable. Glutamate-pyruvate aminotransferase activity was not associated with the proplastid fractions of the non-green tissues. The distribution of glutamate-oxaloacetate aminotransferase was different from that of glutamate-pyruvate aminotransferase. Glutamate-oxaloacetate aminotransferase was found in chloroplasts, proplastids, mitochondria, microbodies and in the supernatant. Evidence is presented that glutamate-pyruvate and glutamate-glyoxylate aminotransferase activities were catalyzed by the same enzyme. Both activities showed the same organelle distribution on sucrose gradients and both were eluted at the same salt concentration from DEAE-cellulose. By chromatography of preparations from rye leaf extracts on DEAE-cellulose two forms of glutamate-pyruvate (glyoxylate) aminotransferase were separated. The major fraction eluting at a low salt concentration was identified as peroxisomal form and the minor fraction eluting at a higher salt concentration was identified as a mitochondrial form. Both the glutamate-glyoxylate and the glutamate-pyruvate aminotransferase activities of the peroxisomal as well as of the mitochondrial forms of the enzyme were strongly (about 80%) inhibited by the presence of 10 mM glycidate, previously described as an inhibitor of glutamate-glyoxylate aminotransferase in tobacco tissue. Pig heart glutamate-pyruvate aminotransferase exhibited no glutamate-glyoxylate aminotransferase activity and was only slightly inhibited by glycidate. The development of glutamate-pyruvate aminotransferase activity in the leaves of rye seedlings was strongly increased in the light, relative to dark-grown seedlings, and very similar to that of catalase activity while the development of glutamate-oxaloacetate aminotransferase was, in close coincidence with the behavior of leaf growth, only slightly enhanced by light. It is discussed that in green leaves an extrachloroplastic synthesis of alanine is of considerable advantage for the metabolic flow during photosynthesis.     

A mutant of Nicotiana sylvestris deficient in serine glyoxylate aminotransferase activity

Summary A photorespiration mutant of Nicotiana sylvestris lacking serine: glyoxylate aminotransferase activity was isolated in the M₂ generation following EMS mutagenesis. Mutants showing chlorosis in air and normal growth in 1% CO₂ were fed [¹⁴C]-2-glycolate to examine the distribution of ¹⁴C among photorespiratory intermediates. Mutant strain NS 349 displayed a 9-fold increase in serine accumulation relative to wild-type controls. Enzyme assays revealed an absence of serine: glyoxylate aminotransferase (SGAT) activity in NS 349, whereas other peroxisomal enzymes were recovered at normal levels. Heterozygous siblings of NS 349 segregating air-sensitive M₃ progeny in a 31 ratio were shown to contain one half the normal level of SGAT activity, indicating that air sensitivity in NS 349 results from a single nuclear recessive mutation eliminating SGAT activity. Since toxicity of the mutation depends on photorespiratory activity, callus cultures of the mutant were initiated and maintained under conditions suppressing the formation of functional plastids. Plantlets regenerated from mutant callus were shown to retain the SGAT deficiency and conditional lethality in air. The utility of photorespiration mutants of tobacco as vehicles for genetic manipulation of ribulose bisphosphate carboxylase/oxygenase at the somatic cell level is discussed.     

Some properties of serine: glyoxylate aminotransferase from rye seedlings (Secale cereale L.).

Serine: glyoxylate aminotransferase (EC 2.6.1.45) from rye seedlings catalysed transamination between L-serine and glyoxylate according to the Ping Pong Bi Bi mechanism with double substrate inhibition. As judged from the Km values, L-serine, L-alanine, and L-asparagine served as substrates for the enzyme with glyoxylate, whereas L-alanine and L-asparagine underwent transamination with hydroxypyruvate as acceptor. Pyridoxal phosphate (PLP) seems to be rather loosely bound to the enzyme protein. Aminooxyacetate and D-serine were found to be pure competitive inhibitors of the enzyme, with Ki values of 0.12 microM and 1.6 mM, respectively. Among the PLP inhibitors isonicotinic acid hydrazide and hydroxylamine were far less effective than aminooxyacetate (20% and 70% inhibition at 0.1 mM concentration, respectively). Inhibition by the SH group inhibitors at 1 mM concentration did not exceed 50%. L-Serine distinctly diminished the inhibitory effect of this type inhibitors. Preincubation of the enzyme with glyoxylate distinctly diminished transamination. Glyoxylate limited the inhibitory action of formaldehyde probably by competing for the reactive groups present in the active centre.     

Photorespiratory toxicity in autotrophic cell cultures of a mutant of Nicotiana sylvestris lacking serine: glyoxylate aminotransferase activity

Procedures were devised for heterotrophic culture and autotrophic establishment of protoplast-derived cell cultures from the sat mutant of Nicotiana sylvestris Speg. et Comes lacking serine: glyoxylate aminotransferase (SGAT; EC 2.6.1.45) activity. Increasing photon flux rates (dark, 40, 80 mol quanta·m^-2·s^-1) enhanced the growth rate of autotrophic (no sucrose) wild-type (WT) cultures in air and 1% CO₂. Mutant cultures showed a similar response to light under conditions suppressing photorespiration (1% CO₂), and maintained 65% of WT chlorophyll levels. In normal air, however, sat cultures developed severe photorespiratory toxicity, displaying a negligible rate of growth and rapid loss of chlorophyll to levels below 1% of WT. Low levels of sucrose (0.3%) completely reversed photorespiratory toxicity of the mutant cells in air. Mutant cultures maintained 75% of WT chlorophyll levels in air, displayed light stimulation of growth, and fixed ¹⁴CO₂ at rates identical to WT. Autotrophic sat cultures accumulated serine to levels nearly nine-fold above that of WT cultures in air. Serine accumulated to similar levels in mixotrophic (0.3% sucrose) sat cultures in air, but had no deleterious effect on fixation of ¹⁴CO₂ or growth, indicating that high levels of serine are not toxic, and that toxicity of the sat mutation probably stems from depletion of intermediates of the Calvin cycle. Autotrophic sat cultures were employed in selection experiments designed to identify spontaneous reversions restoring the capacity for growth in air. From a population of 678 000 sat colonies, 23 plantlets were recovered in which sustained growth in air resulted from reacquisition of SGAT activity. Twenty-two had SGAT levels between 25 and 50% of WT, but one had less than 10% of WT SGAT activity, and eventually developed symptoms typical of the sat mutant. The utility of autotrophic sat cultures for selection of chloroplast mutations diminishing the oxygenase activity of ribulose-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) is discussed.Abbreviations Chl chlorophyll - DW dry weight - FW fresh weight - SGAT Serine:glyoxylate aminotransferase - WT wild-type     

Purification and properties of L-alanine:4,5-dioxovalerate aminotransferase from Chlorella regularis

The enzyme L-alanine:4,5-dioxovalerate aminotransferase (EC 2.6.1.43), which catalyzes the synthesis of 5-aminolevulinic acid, was purified 161-fold from Chlorella regularis. The enzyme also showed L-alanine:glyoxylate aminotransferase activity (EC 2.6.1.44). The activity of glyoxylate aminotransferase was 56-fold greater than that of 4,5-dioxovalerate aminotransferase. The ratio of the two activities remained nearly constant during purification, and when the enzyme was subjected to a variety of treatments. 4,5-Dioxovalerate aminotransferase activity was competitively inhibited by glyoxylate, with a Ki value of 0.5 mM. Double-reciprocal plots of velocity versus 4,5-dioxovalerate with varying L-alanine concentrations indicate a ping-pong reaction mechanism. The apparent Km values for 4,5-dioxovalerate and L-alanine were 0.12 and 3.5 mM, respectively. The enzyme is an acidic protein having an isoelectric point of 4.8. The molecular weight of the enzyme was estimated to be 126,000, with two identical subunits. These results suggest that, in Chlorella, as in bovine liver mitochondria and Euglena, both 4,5-dioxovalerate and glyoxylate aminotransferase activities are associated with the same protein. From the activity ratio of transamination and catalytic properties, it is concluded that this enzyme does not function primarily as a part of the 5-carbon pathway to 5-aminolevulinic acid synthesis.