Cysteine sulfinate transamination activity of aspartate aminotransferases.

Aspartate aminotransferases from pig heart cytosol and mitochondria, $\text{Escherichia coli}$ B and $\text{Pseudomonas striata}$ accepted L-cysteine sulfinate as a good substrate. The mitochondrial isoenzyme and the $\text{Escherichia}$ enzyme showed higher activity toward L-cysteine sulfinate than toward the natural substrates, L-glutamate and L-aspartate. The cytosolic isoenzyme catalyzed the L-cysteine sulfinate transamination at 50% the rate of L-glutamate transamination. The $\text{Pseudomonas}$ enzyme had the same reactivity toward the three substrates. Antisera against the two isoenzymes and the $\text{Escherichia}$ enzyme inactivated almost completely cysteine sulfinate transamination activity in the crude extracts of pig heart muscle and $\text{Escherichia coli}$ B, respectively. These results indicate that cysteine sulfinate transamination is catalyzed by aspartate aminotransferase in these cells.     

Evolutionary analysis of aspartate aminotransferases

Aspartate aminotransferase isoenzymes are located in both the cytosol and organelles of eukaryotes, but all are encoded in the nuclear genome. In the work described here, a phylogenetic analysis was made of aspartate aminotransferases from plants, animals, yeast, and a number of bacteria. This analysis suggested that five distinct branches are present in the aspartate aminotransferase tree. Mitochondrial forms of the enzyme form one distinct group, bacterial aspartate aminotransferase formed another, and the plant and vertebrate cytosolic isoenzymes each formed a distinct group. Plant cytosolic isozymes formed a further group of which the plastid sequences were a member. The yeast mitochondrial and cytosolic aspartate aminotransferases formed groups separate from other members of the family. Correspondence to: C.J. Marshall     

Pyridoxamine-alpha-keto acid transamination activity of aspartate apoaminotransferases

Pyridoxamine-α-keto acid transamination activities of homogeneous aspartate apoaminotransferases from various organisms were determined. Aspartate apoaminotransferases from pig heart cytosol and bakers' yeast utilized both oxalacetate and α-keto-glutarate as amino acceptors, while those from pig heart mitochondria and bacteria (Escherichiacoli B and Pseudomonas striata) showed reactivity only toward oxalacetate. Specific activities of bacterial aspartate apoaminotransferases were very high compared to those of the yeast and animal apoenzymes. Phosphate and various anions, including sulfate, raised the pyridoxamine-α-keto acid transamination activity of all the aspartate apoaminotransferases examined. However, a high concentration of phosphate inhibited the reaction.     

Suicide inactivation of bacterial cystathionine gamma-synthase and methionine gamma-lyase during processing of L-propargylglycine.

L-Propargylglycine, a naturally occurring gamma, delta-acetylenic alpha-amino acid, induces mechanism-based inactivation of two pyridoxal phosphate dependent enzymes of methionine metabolism: (1) cystathionine gamma-synthease, which catalyzes a gamma-replacement reaction in methionine biosynthesis, and (2) methionine gamma-lyase, which catalyzes a gamma-elimination reaction in methionine breakdown. Biphasic pseudo-first-order inactivation kinetics were observed for both enzymes. Complete inactivation is achieved with a minimum molar ratio ([propargylglycine]/[enzyme monomer]) of 4:1 for cystathionine gamma-synthase and of 8:1 for methionine gamma-lyase, consistent with a small number of turnovers per inactivation event. Partitioning ratios were determined directly from observed primary kinetic isotope effects. [alpha-2H]Propargylglycine displays kH/kD values of about 3 on inactivation half-times. [alpha-3H]-Propargylglycine gives release of tritium to solvent nominally stoichiometric with inactivation but, on correction for the calculated tritium isotope discrimination, partition ratios of four and six turnovers per monomer inactivated are indicated for cystathionine gamma-synthase and methionine gamma-lyase, respectively. The inactivation stoichiometry, using [alpha-14C]-propargylglycine, is four labels per tetramer of cystathionine gamma-synthase but usually only two labels per tetramer of methionine gamma-lyase (half-of-the-sites reactivity). Two-dimensional urea isoelectrofocusing/NaDodSO4 electrophoresis suggests (1) that both native enzymes are alpha 2 beta 2 tetramers where the subunits are distinguishable by charge but not by size and (2) that, while each subunit of a cystathionine gamma-synthase tetramer becomes modified by propargylglycine, only one alpha and one beta subunit may be labeled in an inactive alpha 2 beta 2 tetramer of methionine gamma-lyase. Steady-state spectroscopic analyses during inactivation indicated that modified cystathionine gamma-synthase may reprotonate C2 of the enzyme--inactivator adduct, so that the cofactor is still in the pyridoxaldimine oxidation state. Fully inactivated methionine gamma-lyase has lambda max values at 460 and 495 nm, which may represent conjugated pyridoximine paraquinoid that does not reprotonate at C2 of the bound adduct. Either species could arise from Michael-type addition of an enzymic nucleophile to an electrophilic 3,4-allenic paraquinoid intermediate, generated initially by propargylic rearrangement upon a 4,5-acetylenic pyridoximine structure, as originally proposed for propargylglycine inactivation of gamma-cystathionase [Abeles, R., & Walsh, C. (1973) J. Am. Chem. Soc. 95, 6124]. It is reasonable that cystathionine gamma-synthase is the major in vivo target for this natural acetylenic toxin, the growth-inhibitory effects of which are reversed by methionine.     

Inactivation of aspartate aminotransferase during transamination with chloropyruvate. Evidence against modification of Cys390 in cytosolic isoenzyme.

Both the cytosolic and mitochondrial isoenzyme of aspartate aminotransferase from pig heart were inactivated during transamination with chloropyruvate. Inactivation occurred with L-alanine as the amino group donor in the presence of potassium formate. When L-glutamate or L-aspartate was employed as the amino group donor in the transamination reaction with chloropyruvate, no inactivation occurred. This is in contrast to the case of inactivation by bromopyruvate (Okamoto, M. &; Morino, Y. (1973) J. Biol. Chem. $\text{248}$, 82–90) where these natural dicarboxylic amino acid substrates were effective in the transamination reaction leading to syncatalytic inactivation (Birchmeier, W. &; Christen, P. (1974) J. Biol. Chem. $\text{249}$, 6311–6315). The Cys₃₉₀ in the cytosolic isoenzyme which was modified in the syncatalytic inactivation was not modified under the present condition for inactivation with either chloropyruvate or bromopyruvate.     

Effect of trophic conditions on aspartate transamination in wheat plants

The enzymatic transamination reactions between aspartic and α-ketoglutaric acid and between aspartic and pyruvic acid were studied in fresh dialysed extracts of young wheat plants cultivated under various trophical conditions, in mineral solution (Knop), in the solution of an soil organic substance (potassium humate) and without nutrients (H₂O). Simultaneously, the level of endogenic aspartic acid, glutamic acid and the growth values were determined. The enzymatic reactions were characterized by determining the optimum pH, the time course, and the effect of coenzyme and of inhibitors. The activity of the aspartate-glutamate transaminase from the root system of plants was considerably higher than the activity of the overground organs. The enzymatic activity from both parts of the plant was inversely proportional to the growth rate: intensive growth of the plants from the Knop variant was connected with their low enzymatic activity; the level of endogenic glutamic acid was high. The slow growth of the plants without nutrients was connected with a higher enzymatic activity; the level of endogenic glutamic acid was low. The plants from the potassium humate variant had an intermediate position between these two variants from the point of view of growth as well as from that of enzymatic activity. The plants with insufficient nutrition (slow growth, low level of endogenic glutamic acid) apparently have a low capacity for supplementing the glutamic acid deficit, which is essential for the metabolic processes, by increasing the activity of the reactions leading to glutamic acid synthesis (Asp-Glu) and, on the other hand, by decreasing the reactions utilizing it (Glu-Ala). For wheat plants the active aspartate-glutamate reaction is obviously physiologically more important than the direct reaction glutamate-aspartate and the reaction aspartate-alanine which in all cases had a very low activity.     

Reactivity of sulphydryl groups of cytosolic and mitochondrial bovine aspartate aminotransferases

Summary Reactivity of sulphydryl groups of cytosolic and mitochondrial aspartate aminotransferases from ox heart has been studied. A total of 5 and 7 cysteine residues per monomer are present in cAATo and mAATo, respectively. In native conditions only a single sulphydryl group can be titrated by Nbs₂ while the catalytic activity remains unchanged, however in the mitochondrial isozyme the reactivity depends on the functional state of the enzyme. Reactivity toward NEM reveals the existence of a syncatalytic sulphydryl group in the cytosolic isozyme. Titration of cAATo with pMB at pH 8 and pH 5 confirms the existence of two exposed sulphydryl groups with a different reactivity. The results compared with those reported on the corresponding isozymes from pig and chicken heart show that syncatalytic sulphydryl groups are of general occurrence in these enzymes.     

The involvement of aspartate aminotransferases in ammonium assimilation in lupin nodules

Aspartate aminotransferase (AAT) activity has been detected in the plant and bacteroid fractions of lupin nodules, and in free-living Rhizobium lupini. Two electrophoretically distinct forms of AAT were detected in the plant fraction of the nodule and a third form in the bacteroid fraction. AAT activity increased in the plant fraction during nodule development and this increase may be due to an increase in the activity of one of the AAT forms in this fraction. The single form of AAT detected in the bacteroid fraction had the same electrophoretic mobility as that detected in free-living R. lupini. The nodulated roots of lupins, grown in a media supplemented with nitrate and ammonium, had a 3- and 4-fold lower activity of AAT and nitrogenase activity respectively, compared to the nodulated roots of plants grown in the absence of added nitrogen. A role for the plant AAT in ammonium assimilation in lupin nodules is proposed.     

Irreversible inactivation of rat liver tyrosine aminotransferase

Homogenates prepared from rat livers irreversibly inactivate tyrosine aminotransferase, both endogenous and purified exogenous enzyme, in the presence of certain compounds which bind to pyridoxal 5′-P. The rate of inactivation ranged from a half-life of 0.72 to greater than 15 hr. The pyridoxal 5′-P binding compounds may be considered to be structural analogs for α-ketoglutarate or l-tyrosine, both of which are substrates for the enzyme. l-Cysteine and l-DOPA are the most effective compounds tested of each of the two structural analog classes, respectively. Absence of the carboxyl group from l-cysteine or l-DOPA has little effect on the half-life of the enzyme, whereas absence or substitution of the amino group results in an increased enzyme half-life. Absence of the —SH group from l-cysteine or of the 3′-OH group from l-DOPA results in little or no inactivation of the enzyme ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ increased to greater than 15 hr). Semicarbazide and hydroxylamine have little effect on the stability of the enzyme. Addition of pyridoxal 5′-P to homogenates incubated with l-cysteine or l-DOPA inhibits the inactivation of the enzyme. However, the addition of cofactor to inactivated enzyme does not restore lost activity.There is a disappearance of antigenic cross-reacting material during inactivation of the enzyme. This loss of specific cross-reacting material occurs at a slower rate than the loss of enzyme activity, indicating that enzymatic activity is lost prior to loss of antigenic recognition. A three-step proposal is presented to explain the data observed in which the first step is a reversible loss of pyridoxal 5′-P from the enzyme, followed by a specific irreversible inactivation of the enzyme, and ending with nonspecific proteolysis or degradation of the inactivated enzyme molecules.     

Partial purification and characterization of aspartate aminotransferases from seedling oat leaves.

As relatively little information is available on the properties of aspartate aminotransferase from photosynthetic tissue, isolation and characterization of the two major electrophoretically distinct forms of this enzyme from seedling oat leaf homogenates were undertaken. These two forms are designated I for the more anionic form and II for the less anionic form. Form I, 80 to 90% of the total activity, has been purified to a specific activity of 120 mumol/min/mg of protein (1100-fold) and is estimated to be 90 to 95% homogeneous, as judged by analytical polyacrylamide gel electrophoresis. Form II, 10 to 20% of the total activity, has been purified to a specific activity of approximately 6 mumol/min/mg of protein (300-fold). Both forms exhibit optimal activity at pH 7.5. Michaelis constants do not differ greatly between forms I and II and are similar to those reported for the pig heart cytosolic enzyme as well as aspartate aminotransferase from other plant sources. A molecular weight of 130,000 for the purified aspartate aminotransferase I was estimated by sedimentation equilibrium centrifugation; molecular weights of the two forms are similar as estimated by sucrose density gradient centrifugation. No activation by pyridoxal phosphate has been observed during purification.     

Multispecific aspartate and aromatic amino acid aminotransferases in Escherichia coli.

Two aminotransferases from Escherichia coli were purified to homogeneity by the criterion of gel electrophoresis. The first (enzyme A) is active on L-aspartic acid, L-tyrosine, L-phenylalanine, and L-tryptophan; the second (enzyme B) is active on the aromatic amiono acids. Enzyme A is identical in substrate specificity with transaminase A and is mainly an aspartate aminotransferase; enzyme B has never been described before and is an aromatic amino acid aminotransferase. The two enzymes are different in the Vmax and Km values with their common substrates and pyridoxal phosphate, in heat stability (enzyme A being heat-stable and enzyme B being heat-labile at 55 degrees) and in pH optima with the amino acid substrates. They are similar in their amino acid composition, each enzyme appears to consist of two subunits, and enzyme B may be converted to enzyme A by controlled proteolysis with subtilsin. The conversion was detected by the generation of new aspartate aminotransferase activity from enzyme B and was further verified by identification by acrylamide gel electrophoresis of the newly formed enzyme A. The two enzymes appear to be products of two genes different in a small, probably terminal, nucleotide sequence.