Mendel Mazelis

The enzymology of lysine biosynthesis in higher plants: complete localization of the regulatory enzyme dihydrodipicolinate synthase in the chloroplasts of spinach leaves.

Lysine is synthesized in higher plants from aspartate, via the diaminopimelate pathway [ 11. Intact isolated chloroplasts have been shown to synthesize all the aspartatederlved amino acids, including lysine, from exogenous [ 14C] aspartate in the light [ 21. The final enzyme of lysine biosynthesis, diaminopimelate decarboxylase (EC 4.1 .1.20) has been found in Vicia fuba chloroplasts [3] and shown to be completely localized in the chloroplasts in pea (Pisum sutivum) leaves [4]. A number of other enzymes of the aspartate pathway have also been found in chloroplasts (reviewed [5]). In most instances the complete intracellular distributions have not been determined. The first enzyme unique to lysine synthesis is dihydrodipicolinate synthase (EC 4.2.1.52), and this enzyme has been found in a variety of plant tissues [ 61. So far there has been no report of the localization of this enzyme, though the results in [2] suggest that it is at least partly, if not completely, located in the chloroplast . As part of our investigation of the properties of spinach leaf dlhydrodipicolinate synthase we have studied the distribution of the enzyme in mechanically prepared leaf homogenates and in protoplasts. This report clearly shows the chloroplast contains all of the enzyme present in the spinach leaf. Some of the difficulties and problems associated with the customary enzyme assay are also described as well as ameliorative modifications.

Conversion of ornithine into proline by enzymes from germinating peanut cotyledons

Abstract During germination a marked synthesis of proline occurs in the cotyledons of peanuts. A soluble enzyme system which converted ornithine into proline was extracted from acetone powders of 3–5-day-old peanut cotyledons. The reaction required ornithine, α-ketoglutarate, and a reduced pyridine nucleotide. NADPH was much more effect than NADH. An l -ornithine:2-oxoacid aminotransferase (EC 2.6.1.13) has been purified about six-fold from the extracts. The reaction produces a pyrroline carboxylate as indicated by a positive reaction with o-aminobenzaldehyde. An optimum pH of 8·0 was found with tris-HCl. The Km for α-ketoglutarate was 2·5 mM and for l -ornithine 5·0 mM. Exogenous pyridoxal phosphate was not required, but 0·1 mM hydroxylamine inhibited the reaction by 90 per cent. α-Ketoglutarate was quite specific as the acceptor, pyruvate, oxaloacetate, and glyoxylate showing very little activity. Fractionation of germinating cotyledon homogenates showed that the mitochondrial fraction had a specific activity more than eight-fold that in the remaining soluble cytoplasm, but the total soluble activity was six times that of the mitochondrial fraction.

The C-S lyases of higher plants: Preparation and properties of homogeneous alliin lyase from garlic (Allium sativum)

Alliin lyase from garlic (Allium sativum) has been purified to homogeneity. The purification procedure involves the use of affinity chromatography on concanavalin A-Sepharose 4B. Addition of polyvinylpolypyrrolidone to the homogenizing medium greatly improves the specific activity of the extract. The enzyme is a glycoprotein as seen by its ability to bind to concanavalin A-Sepharose 4B and by its positive periodic acid-Schiff base stain. It has a carbohydrate content of 5.5%. Km values for this enzyme were estimated to be 5.7 mM for S-ethyl-L-cysteine sulfoxide and 3.3 mM for S-allyl-L-cysteine sulfoxide. The molecular weight of this garlic enzyme, as determined by gel filtration, was found to be 85,000; the molecule consists of two equal subunits of Mr 42,000. The amino acid content was found to be similar to that reported previously for onion alliin lyase, although there is twice as much tryptophan in the garlic alliin lyase as in the onion enzyme. By both chemical and spectral methods the enzyme was found to have two molecules of pyridoxal 5-phosphate per enzyme molecule, suggesting one per subunit. There are significant differences in the nature of these findings from those previously reported from this laboratory for the onion enzyme. Studies are in progress to compare further the alliin lyases from garlic and onion.

Demonstration and characterization of cysteine sulfoxide lyase in the cruciferae

Abstract An enzyme which degrades cysteine sulfoxides has been found in members of the genus Brassica of the family Cruciferae. The name 3-alkylsulphinylalanine alkyl sulphenate lyase (deaminating) (cysteine sulfoxide lyase) is proposed for this enzyme. 1 The enzyme has been purified about eleven-fold from broccoli buds. The broccoli lyase is similar in its mode of action to alliinase which has been described in Allium species. The pH optimum of the broccoli enzyme is between 8·4 and 8·6 in borate buffer. The products of the reaction are pyruvate, ammonia, and alkyl alkane thiosulfinates. The Km is 2·7 × 10 −3 M with l -methylsulphinylalanine as the substrate. Studies with acetone powders and aged preparations, and inhibition studies indicate that pyridoxal-5′-phosphate is a coenzyme. s -methyl- l -cysteine was not a substrate, nor did it inhibit the reaction when present in equimolar concentration with the sulfoxide. Equimolar amounts of l -cysteic acid and l -cysteine sulfinic acid resulted in inhibitions of 30–40 per cent in the utilization of l -methylsulphinylalanine.

The enzymology of lysine biosynthesis in higher plants. The occurrence, characterization and some regulatory properties of dihydrodipicolinate synthase.

Lysine, an essential amino acid for human nutrition, is frequently the limiting amino acid in plant protein in terms of nutritional quality. It is the limiting amino acid in all the important cereal grains [ 1]. The biosynthesis of this amino acid in higher plants and its regulation are of considerable practical importance. In vivo studies of the incorporation of radioactive precursors into lysine indicate that higher plants utilize the diaminopimelate pathway [2,3]. For definitive proof of the existence of this route, the individual enzymes for each step must be demonstrated. Diaminopimelate decarboxylase (EC 4.1.1.20) which catalyses the final step in the sequence, has been found in a large number of higher plant tissues [4 -7] . It is the only enzyme in the pathway which has been extensively characterized [4 -7] . In addition the presence of dihydrodipicolinate synthase (EC 4.2.1.52) in maize seedlings has been reported [8]. This enzyme catalyses the first step in the pathway, the condensation of ASA and pyruvate to dihydrodipicolinate. The occurrence of dihydrodipicolinate synthase in a number of higher plants is now demonstrated and

Spinach leaf dihydrodipicolinate synthase: Partial purification and characterization

Abstract The first enzyme unique to lysine biosynthesis in higher plants, dihydrodipicolinate synthase, has been partially purified from spinach leaves, using ion exchange chromatography, hydrophobic interaction chromatography and gel filtration. The spinach enzyme is moderately stable to short-term exposure to heat, in contrast to the pea leaf enzyme, but is unstable on storage even at −20°. Thiol reagents interfere with the calorimetric assay used, and so cannot be routinely used to stabilize the enzyme, which has an active sulphydryl group. The MW of the enzyme is 115000 (gel filtration). Lysine is a potent inhibitor with an I (0.5) of 2OμM, whilst the lysine analogue S-β-aminoethylcysteinc has an I (0.5) of 400 μM. The Kt´ m for aspartic-β-semialdehyde was determined to be 1.4mM, but this compound demonstrated marked substrate inhibition at concentrations above 7 mM, increasing the apparent S (0.5) for the second substrate, pyruvate.

Alliin lyase: Preparation and characterization of the homogeneous enzyme from onion bulbs

Abstract Alliin lyase (alliin alkyl-sulfenate-lyase, EC 4.4.1.4; alliinase) of onion bulbs has been purified to homogeneity. The enzyme catalyzes the following β-elimination reaction. Based on sedimentation equilibrium centrifugation data, the enzyme has a molecular weight of 150,000. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single subunit of M r 50,000. Urea-polyacrylamide gel electrophoresis also yielded a single band after staining with Coomassie blue. The enzyme was shown to be a glycoprotein by the use of a periodic acid-Schiff base staining technique on SDS-PAGE-treated preparations. The carbohydrate moiety was 5.8% of the total protein molecular weight. It consisted of simple sugars, hexoseamines, and methyl pentose, but no sialic acid was found. The enzyme activity showed no requirement for exogenous pyridoxal 5′-phosphate. Inhibition and spectrophotometric studies indicated this cofactor was already bound to the enzyme. Chemical analysis revealed that there were 3 mol of pyridoxal 5′-phosphate per 150,000 g of enzyme.

Cleavage of l-cystine by soluble enzyme preparations from Brassica species

Abstract The decomposition of l -cystine to pyruvate and the persulfide, thiocysteine, is catalyzed by soluble enzymes obtained from tissues of brassica species. the enzyme, called trivially cystine lyase, requires pyridoxal-5′-PO4 for activity; however, the stimulation of activity by exogenous cofactor is dependent on the species. Partial purification of the enzyme from the root of B. napobrassica (rutabaga) was accomplished. The rutabaga lyase was completely inactive in the absence of added pyridoxal-5′-PO4. The PH optimum was between 8.5 And 8.9. The lyase was specific for the l -configuration of the amino acid and was only slightly inhibited by equimolar concentrations of The D -Form. The km for l -cystine was 1 mm and was 0.5 μm for pyridoxal-5′-PO4. There was a severe competitive inhibition by thiol compounds such as cysteine and reduced glutathione. The ki for both of these compounds was 1.5 × 10−4 m . The enzyme preparation was capable of utilizing s-Methyl- l -cysteine sulfoxide and l -cysteine-s-Sulfate as substrates. It was completely inactive toward l -cystathionine and dl -allocystathionine. The stoichiometry of the reaction was one mole of thiocysteine and one mole of pyruvate produced per mole of cystine.

Decomposition of methyl methionine sulfonium salts by a bacterial enzyme

Summary 1. A soil microorganism which can utilize methyl methionine sulfonium salts as the sole source of C, N and S has been isolated by enrichment culture. An enzyme has been partially purified from sonic extracts of the bacterium which degrades methyl methionine sulfonium to dimethyl sulfide and homoserine. 2. The pH optimum for the breakdown is 7.8. The enzyme is specific for the L -form of the methionine derivative. The Km for the L -form is 1.5·10−3 M. The D -form does not inhibit the reaction. The addition of pyridoxal 5′-phosphate does not stimulate the reaction. Hydroxylamine, NaCN, isonicotinic acid hydrazide, and p-hydroxymercuribenzoate do not inhibit the enzyme. Dimethylacetothetin, dimethyl-β-propiothetin and methionine sulfoxide are not substrates, however, (−)S-adenosyl- L -methionine is degraded by the enzyme preparation.

The metabolism of methionine in higher plants.: Catabolism of the methyl group by seedlings of various species

Abstract l -Methionine- 14 CH 3 was rapidly metabolized after infiltration into pea, pumpkin, kohlrabi, and sesbania seedlings. In all cases the labeled carton atom was incorporated into the organic acid, neutral sugar, and amino acid fractions prepared from the seedlings after incubation with the radioactive methionine. Up to 4 per cent of the total activity fed was recovered as 14 CO 2 after 24 hr. At this time as much as 30 per cent of the label was found in the organic acid fraction. In sesbania the activity in this fraction was localized in one compound primarily, whereas the other plants appeared to have two predominant products formed. Among the labeled components of the amino acid fraction, methionine, methionine sulfoxide, serine, and methyl methionine sulfonium were identified and were found in every species tested. The formation of methionine sulfoxide appeared to be a physiological process and not an artifact of the isolation procedure. The presence of the methyl sulfonium salt of methionine in every case implies that this compound may have a significant metabolic role in higher plants.