(1-->3)-beta-d-Glucan Synthase from Sugar Beet : II. Product Inhibition by UDP

The mode of inhibition of UDP, one of the products of the reaction catalyzed by (1→3)-β-d-glucan synthase in sugar beet (Beta vulgaris L.) was investigated. In the absence of added UDP, the enzyme, in the presence of Ca²⁺, Mg²⁺, and cellobiose, exhibited Michaelis-Menten kinetics and had an apparent K_m of 260 micromolar for UDP-glucose. Complex effects on the kinetics of the (1→3)-β-d-glucan synthase were observed in the presence of UDP. At high UDP-glucose concentrations, i.e. greater than the apparent K_m, UDP behaved as a competitive inhibitor with an apparent K_i of 80 micromolar. However, at low UDP-glucose concentrations, reciprocal plots of enzyme activity versus substrate concentration deviated sharply from linearity. This unusual effect of UDP is similar to that reported for fungal (1→3)-β-d-glucan synthase. However, papulacandin B, a potent inhibitor of this fungal enzyme, had no effect on the plant (1→3)-β-d-glucan synthase isolated from sugar beet petioles. The inhibitory effect of UDP was also compared with other known inhibitors of glucan synthases.     

Nucleoside Diphosphate-sugar 4-Epimerases I. Uridine Diphosphate Glucose 4-Epimerase of Wheat Germ

Uridine diphosphate (UDP)-glucose 4-epimerase (EC 5.1.3.2) has been purified over 1000-fold from extracts of wheat germ by MnCl₂ treatment, (NH₄)₂SO₄ fractionation, Sephadex column chromatography, and adsorption onto and elution from calcium phosphate gel. The enzyme has a pH optimum of 9.0. Km values are 0.1 mm for UDP-d-galactose and 0.2 mm for UDP-d-glucose. NAD is required for activity; K_a = 0.04 mm. NADH is an inhibitor strictly competitive with NAD; K_i = 2 μm. Wheat germ also contains UDP-l-arabinose 4-epimerase (EC 5.1.3.5) and thymidine diphosphate (TDP)-glucose 4-epimerase which are distinct from UDP-glucose 4-epimerase.     

Enzymatic Biosynthesis of Novel Resveratrol Glucoside and Glycoside Derivatives

A UDP glucosyltransferase from Bacillus licheniformis was overexpressed, purified, and incubated with nucleotide diphosphate (NDP) d- and l-sugars to produce glucose, galactose, 2-deoxyglucose, viosamine, rhamnose, and fucose sugar-conjugated resveratrol glycosides. Significantly higher (90%) bioconversion of resveratrol was achieved with α-d-glucose as the sugar donor to produce four different glucosides of resveratrol: resveratrol 3-O-β-d-glucoside, resveratrol 4′-O-β-d-glucoside, resveratrol 3,5-O-β-d-diglucoside, and resveratrol 3,5,4′-O-β-d-triglucoside. The conversion rates and numbers of products formed were found to vary with the other NDP sugar donors. Resveratrol 3-O-β-d-2-deoxyglucoside and resveratrol 3,5-O-β-d-di-2-deoxyglucoside were found to be produced using TDP-2-deoxyglucose as a donor; however, the monoglycosides resveratrol 4′-O-β-d-galactoside, resveratrol 4′-O-β-d-viosaminoside, resveratrol 3-O-β-l-rhamnoside, and resveratrol 3-O-β-l-fucoside were produced from the respective sugar donors. Altogether, 10 diverse glycoside derivatives of the medically important resveratrol were generated, demonstrating the capacity of YjiC to produce structurally diverse resveratrol glycosides.     

Partial Purification and Properties of l-Glutamine d-Fructose 6-Phosphate Amidotransferase from Phaseolus aureus

l-Glutamine d-fructose 6-phosphate amidotransferase (EC 2.6.1.16) was extracted and purified 600-fold by acetone fractionation and diethylaminoethyl cellulose column chromatography from mung bean seeds (Phaseolus aureus). The partially purified enzyme was highly specific for l-glutamine as an amide nitrogen donor, and l-asparagine could not replace it. The enzyme showed a pH optimum in the range of 6.2 to 6.7 in phosphate buffer. Km values of 3.8 mm and 0.5 mm were obtained for d-fructose 6-phosphate and l-glutamine, respectively. The enzyme was competitively inhibited with respect to d-fructose 6-phosphate by uridine diphosphate-N-acetyl-d-glucosamine which had a Ki value of 13 μm. Upon removal of l-glutamine and its replacement by d-fructose 6-phosphate and storage over liquid nitrogen, the enzyme was completely desensitized to inhibition by uridine diphosphate-N-acetyl-d-glucosamine. This indicates that the inhibitor site is distinct from the catalytic site and that uridine diphosphate-N-acetyl-d-glucosamine acts as a feedback inhibitor of the enzyme.     

Uridylate trapping, induction of UTP deficiency, and stimulation of pyrimidine synthesis de novo by d-galactosone

d-Galactosone (d-lyxo-2-hexosulose) is phosphorylated and metabolized to the uridine diphosphate derivative in AS-30D hepatoma cells and rat liver. These reactions were catalysed in vitro by galactokinase and hexose-1-phosphate uridylyltransferase. Nucleotide analyses by high-performance liquid chromatography and enzymic assays revealed that this galactose analogue interferes with cellular pyrimidine nucleotide metabolism leading to a deficiency of UTP. [¹⁴C]Uridine labelling of hepatoma cells indicated a division of [¹⁴C]uridylate from UTP into UDP-galactosone; the latter was formed at a rate of more than 1.7mmol×h⁻¹×(kg AS-30D or liver wet wt.)⁻¹. As a consequence of UTP deficiency, d-galactosone (1mmol/1 or 1mmol/kg body wt.) strongly enhanced the rate of pyrimidine synthesis de novo as evidenced by incorporation of ¹⁴CO₂ into uridylate and by an expansion of the uridylate pool. This resulted in a doubling of the total acid-soluble uridylate pool within 70min in the hepatoma cells and within 110min in rat liver. Combined treatment of hepatoma cells with d-galactosone and N-(phosphonoacetyl)-l-aspartate, an inhibitor of aspartate carbamoyltransferase, prevented the expansion of the uridylate pool and led to a synergistic reduction of UTP to 10% of the content in control cells. Hepatic UTP deficiency was selective with respect to other nucleotide 5′-triphosphates but was associated with reduced contents of UDP-glucose, UDP-glucuronate, and UDP-N-acetylhexosamines. Isolation of the UDP derivative of d-galactosone revealed an extremely alkali-labile UDP-sugar, probably an isomerization product of UDP-galactosone, that was degraded by elimination of UDP with a half-life of 45min at pH7.5 and 37°C. The instability of UDP-galactosone may contribute in vivo to limit the time period of severe uridine phosphate deficiency in addition to the compensatory role of pyrimidine synthesis de novo. During the initial time period, however, d-galactosone is effective as a powerful uridylate-trapping sugar analogue.     

Purification and Characterization of a Novel Galloyltransferase Involved in Catechin Galloylation in the Tea Plant (Camellia sinensis)

Catechins (flavan-3-ols), the most important secondary metabolites in the tea plant, have positive effects on human health and are crucial in defense against pathogens of the tea plant. The aim of this study was to elucidate the biosynthetic pathway of galloylated catechins in the tea plant. The results suggested that galloylated catechins were biosynthesized via 1-O-glucose ester-dependent two-step reactions by acyltransferases, which involved two enzymes, UDP-glucose:galloyl-1-O-β-d-glucosyltransferase (UGGT) and a newly discovered enzyme, epicatechin:1-O-galloyl-β-d-glucose O-galloyltransferase (ECGT). In the first reaction, the galloylated acyl donor β-glucogallin was biosynthesized by UGGT from gallic acid and uridine diphosphate glucose. In the second reaction, galloylated catechins were produced by ECGT catalysis from β-glucogallin and 2,3-cis-flavan-3-ol. 2,3-cis-Flavan-3-ol and 1-O-galloyl-β-d-glucose were appropriate substrates of ECGT rather than 2,3-trans-flavan-3-ol and 1,2,3,4,6-pentagalloylglucose. Purification by more than 1641-fold to apparent homogeneity yielded ECGT with an estimated molecular mass of 241 to 121 kDa by gel filtration. Enzyme activity and SDS-PAGE analysis indicated that the native ECGT might be a dimer, trimer, or tetramer of 60- and/or 58-kDa monomers, and these monomers represent a heterodimer consisting of pairs of 36- or 34- of and 28-kDa subunits. MALDI-TOF-TOF MS showed that the protein SCPL1199 was identified. Epigallocatechin and epicatechin exhibited higher substrate affinities than β-glucogallin. ECGT had an optimum temperature of 30 °C and maximal reaction rates between pH 4.0 and 6.0. The enzyme reaction was inhibited dramatically by phenylmethylsulfonyl fluoride, HgCl₂, and sodium deoxycholate.     

Inhibition of purine phosphoribosyltransferases of Ehrlich ascites-tumour cells by 6-mercaptopurine

1. The formation of adenosine 5′-phosphate, guanosine 5′-phosphate and inosine 5′-phosphate from [8-¹⁴C]adenine, [8-¹⁴C]guanine and [8-¹⁴C]hypoxanthine respectively in the presence of 5-phosphoribosyl pyrophosphate and an extract from Ehrlich ascites-tumour cells was assayed by a method involving liquid-scintillation counting of the radioactive nucleotides on diethylaminoethylcellulose paper. The results obtained with guanine were confirmed by a spectrophotometric assay which was also used to assay the conversion of 6-mercaptopurine and 5-phosphoribosyl pyrophosphate into 6-thioinosine 5′-phosphate in the presence of 6-mercaptopurine phosphoribosyltransferase from these cells. 2. At pH 7·8 and 25° the Michaelis constants for adenine, guanine and hypoxanthine were 0·9 μm, 2·9 μm and 11·0 μm in the assay with radioactive purines; the Michaelis constant for guanine in the spectrophotometric assay was 2·6 μm. At pH 7·9 the Michaelis constant for 6-mercaptopurine was 10·9 μm. 3. 25 μm-6-Mercaptopurine did not inhibit adenine phosphoribosyltransferase. 6-Mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 4·7 μm) and hypoxanthine phosphoribosyltransferase (K_i 8·3 μm). Hypoxanthine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 3·4 μm). 4. Differences in kinetic parameters and in the distribution of phosphoribosyltransferase activities after electrophoresis in starch gel indicate that different enzymes are involved in the conversion of adenine, guanine and hypoxanthine into their nucleotides. 5. From the low values of K_i for 6-mercaptopurine, and from published evidence that ascites-tumour cells require supplies of purines from the host tissues, it is likely that inhibition of hypoxanthine and guanine phosphoribosyltransferases by free 6-mercaptopurine is involved in the biological activity of this drug.     

Purification and Characterization of Sucrose Synthase from the Cotyledons of Vicia faba L

Partial purification (approximately 270-fold) of sucrose synthase (EC 2.4.1.13) from developing cotyledons of Vicia faba L. cv Maris Bead was achieved by ammonium sulfate fractionation and hydrophobic, affinity, anion-exchange, and gel filtration chromatography. Further purification to homogeneity resulted from gel elution of single bands from native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was identified as a homotetramer with a total molecular mass of 360 kD and subunits of 92 to 93 kD. Antibodies were raised to both native and denatured protein. The identity of the polypeptide was confirmed in western blots using antibodies raised against soybean nodule sucrose synthase. The enzyme has a pH optimum of 6.4 (cleavage direction) and an isoelectric point of 5.5. The affinity of the enzyme for sucrose (K_m) was estimated at 169 mm, and for UDP at 0.2 mm. With uridine diphosphate as the nucleoside diphosphate, the V_max is 4-fold higher than with adenosine diphosphate. Fructose acts as a competitive inhibitor with an inhibitor constant (K_i) of 2.48 mm.     

Stable changes to calcium fluxes in mitochondria isolated from rat livers perfused with α-adrenergic agonists and with glucagon

1. Human uterine cervical stroma was found to contain a Ca²⁺-independent neutral proteinase against casein and N-benzoyl-dl-arginine p-nitroanilide (Bz-dl-Arg-Nan). This enzyme was tightly bound to an insoluble material (20000g pellet) and was solubilized by high concentrations of NaCl or KCl. High concentrations of them in the reaction system, however, inhibited reversibly the activity of this enzyme. 2. The neutral proteinase was partially purified by extraction with NaCl, gel filtration on Sephadex G-200 and affinity chromatography on casein–Sepharose. 3. The optimal pH of this partially purified enzyme was 7.4–8.0 against casein and Bz-dl-Arg-Nan. The molecular weight of the enzyme was found to be about 1.4×10⁵ by gel filtration on Sephadex G-200. 4. The enzyme was significantly inhibited by di-isopropyl phosphorofluoridate (0.1mm). High concentration of phenylmethanesulphonyl fluoride (5mm), 7-amino-1-chloro-3-l-tosylamidoheptan-2-one (0.5mm), antipain (10μm) or leupeptin (10μm) was also found to be inhibitory, but chymostatin (40μg/ml), soya-bean trypsin inhibitor (2.5mg/ml), human plasma (10%, v/v), p-chloromercuribenzoate (1mm), EDTA (10mm) and 1-chloro-4-phenyl-3-l-tosylamidobutan-2-one (1mm) had no effect on the enzyme. 5. The neutral proteinase hydrolysed casein, Bz-dl-Arg-Nan and heat-denatured collagen, but was inactive towards native collagen and several synthetic substrates, such as 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg, 3-carboxypropionyl-Ala-Ala-Ala p-nitroanilide and 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, and also proteoglycan. The enzyme did not act as a plasminogen activator. 6. These properties suggested that a neutral proteinase in the human uterine cervix was different from enzymes previously reported.     

Identification,Purification, and Characterization of a Novel Amino Acid Racemase,Isoleucine 2-Epimerase,from Lactobacillus Species

Accumulation of d-leucine, d-allo-isoleucine, and d-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative γ-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative γ-aminobutyrate aminotransferase gene from L. buchneri JCM 1115 was expressed in recombinant Escherichia coli and then purified to homogeneity. The enzyme catalyzed the racemization of a broad spectrum of nonpolar amino acids. In particular, it catalyzed at high rates the epimerization of l-isoleucine to d-allo-isoleucine and d-allo-isoleucine to l-isoleucine. In contrast, the enzyme showed no γ-aminobutyrate aminotransferase activity. The relative molecular masses of the subunit and native enzyme were estimated to be about 49 kDa and 200 kDa, respectively, indicating that the enzyme was composed of four subunits of equal molecular masses. The K_m and V_max values of the enzyme for l-isoleucine were 5.00 mM and 153 μmol·min⁻¹·mg⁻¹, respectively, and those for d-allo-isoleucine were 13.2 mM and 286 μmol·min⁻¹·mg⁻¹, respectively. Hydroxylamine and other inhibitors of pyridoxal 5′-phosphate-dependent enzymes completely blocked the enzyme activity, indicating the enzyme requires pyridoxal 5′-phosphate as a coenzyme. This is the first evidence of an amino acid racemase that specifically catalyzes racemization of nonpolar amino acids at the C-2 position.     

Properties of pea seedling uracil phosphoribosyltransferase and its distribution in other plants

A uracil phosphoribosyltransferase (UMP-pyrophosphorylase) was found in several angiosperms and was partially purified from epicotyls of pea (Pisum sativum L. cv. Alaska) seedlings. Its pH optimum was about 8.5; its required approximately 0.3 mm MgCl₂ for maximum activity but was inhibited by MnCl₂; its molecular weight determined by chromatography on Sephadex G-150 columns was approximately 100,000; its K_m values for uracil and 5-phosphorylribose 1-pyrophosphate were 0.7 μm and 11 μm; and it was partially resolved from a similar phosphoribosyltransferase converting orotic acid to orotodine 5′-phosphate. Enzyme fractions containing both uracil phosphoribosyl transferase and orotate phosphoribosyltransferase converted 6-azauracil and 5-fluorouracil to products with chromatographic properties of 6-azauradine 5′-phosphate and 5-fluorouridine 5′-phosphate. Uracil phosphoribosyltransferase probably functions in salvage of uracil for synthesis of pyrimidine nucleotides.     

In Vitro Analysis of the H-Hexose Symporter on the Plasma Membrane of Sugarbeets (Beta vulgaris L.)

The mechanism of hexose transport into plasma membrane vesicles isolated from mature sugarbeet leaves (Beta vulgaris L.) was investigated. The initial rate of glucose uptake into the vesicles was stimulated approximately fivefold by imposing a transmembrane pH gradient (ΔpH), alkaline inside, and approximately fourfold by a negative membrane potential (ΔΨ), generated as a K⁺-diffusion potential, negative inside. The -fold stimulation was directly related to the relative ΔpH or ΔΨ gradient imposed, which were determined by the uptake of acetate or tetraphenylphosphonium, respectively. ΔΨ- and ΔpH-dependent glucose uptake showed saturation kinetics with a K_m of 286 micromolar for glucose. Other hexose molecules (e.g. 2-deoxy-d-glucose, 3-O-methyl-d-glucose, and d-mannose) were also accumulated into plasma membrane vesicles in a ΔpH-dependent manner. Inhibition constants of a number of compounds for glucose uptake were determined. Effective inhibitors of glucose uptake included: 3-O-methyl-d-glucose, 5-thio-d-glucose, d-fructose, d-galactose, and d-mannose, but not 1-O-methyl-d-glucose, d- and l-xylose, l-glucose, d-ribose, and l-sorbose. Under all conditions of proton motive force magnitude and glucose and sucrose concentration tested, there was no effect of sucrose on glucose uptake. Thus, hexose transport on the sugarbeet leaf plasma membrane was by a H⁺-hexose symporter, and the carrier and possibly the energy source were not shared by the plasma membrane H⁺-sucrose symporter.     

Purification and some properties of two polyphenol oxidases from bartlett pears

Two polyphenol oxidases (enzymes A and B) from Bartlett pear (Pyrus communis) peelings were purified to electrophoretic homogeneity according to polyacrylamide gel by a combination of Sephadex gel filtration, diethylaminoethyl cellulose chromatography and hydroxyl apatite chromatography. While the two enzymes differ electrophoretically at pH 9.3, chromatographically on hydroxyl apatite, and in the effect of ionic strength on activity, they are similar with respect to chromatography on diethylaminoethyl cellulose, substrate specificity, pH activity relations, inhibition by p-coumaric and benzoic acids, and heat stability. The two enzymes are o-diphenol oxidases with no detectable monophenolase or laccase activities. Pyrocatechol, 4-methyl catechol, chlorogenic acid, and d-catechin are good substrates of the enzymes with K_m values in the range of 2 to 20 mm. Dependences of activity on oxygen and chlorogenic acid concentrations indicate a sequential mechanism for binding of these substrates to enzyme B. V_max and K_m values for oxygen and chlorogenic acid were 103 μmoles O₂ uptake per minute per milligram of enzyme, 0.11 mm and 7.2 mm, respectively, for enzyme B at pH 4.0. Both enzymes had maximum activity at pH 4.0 on chlorogenic acid. K_m values for chlorogenic acid were independent of pH from 3 to 7; the V_max values for both enzymes gave bell-shaped curves as a function of pH. p-Coumaric acid is a simple, linear noncompetitive inhibitor with respect to chlorogenic acid at pH 6.2 with K_i values of 0.38 and 0.50 mm for enzymes A and B, respectively. Benzoic acid is a linear competitive inhibitor with respect to chlorogenic acid at pH 4.0 with K_i values of 0.04 and 0.11 mm for enzymes A and B, respectively.     

Biosynthesis of l-tyrosine O-sulphate from the methyl and ethyl esters of l-tyrosine

1. Rat-liver supernatant preparations are capable of achieving the biological sulphation of l-tyrosine methyl ester, the reaction proceeding maximally at a substrate concentration of 30 mm and at pH 7·0. 2. Two sulphated products are formed, one of which has been identified as l-tyrosine O-sulphate. On the basis of indirect evidence the other product can be assumed to be l-tyrosine O-sulphate methyl ester. 3. An enzyme present in rat-liver supernatant preparations is capable of converting l-tyrosine O-sulphate methyl ester into l-tyrosine O-sulphate. This enzyme is inhibited by l-tyrosine methyl ester. 4. l-Tyrosine ethyl ester also yields two sulphated products when used as an acceptor in the liver sulphating system. One of these has been identified chromatographically as l-tyrosine O-sulphate and the other may be presumed to be l-tyrosine O-sulphate ethyl ester.     

The enzymes forming isopentenyl pyrophosphate from 5-phosphomevalonate (mevalonate 5-phosphate) in the latex of Hevea brasiliensis

1. Phosphomevalonate kinase and 5-pyrophosphomevalonate decarboxylase have been purified from the freeze-dried latex serum of the commercial rubber tree Hevea brasiliensis. 2. The phosphomevalonate kinase was acid- and heat-labile and required the presence of a thiol to maintain activity. 3. The 5-pyrophosphomevalonate decarboxylase was relatively acid-stable and more heat-stable than the phosphokinase. 4. Maximum activity of the phosphokinase was achieved at pH 7.2 with 0.2mm-5-phosphomevalonate (K_m 0.042mm), 2.0mm-ATP (K_m 0.19mm) and 8mm-Mg²⁺ at 40°C. The apparent activation energy was 14.8kcal/mol. 5. Maximum activity of 5-pyrophosphomevalonate decarboxylase was achieved at pH5.5–6.5 with 0.1mm-5-pyrophosphomevalonate (K_m 0.004mm), 1.5mm-ATP (K_m 0.12mm) and 2mm-Mg²⁺. The apparent activation energy was 13.7kcal/mol. The enzyme was somewhat sensitive to inhibition by its products, isopentenyl pyrophosphate and ADP.     

The regulation of glyceride synthesis in isolated white-fat cells. The effects of palmitate and lipolytic agents

1. 0.5mm-Palmitate stimulated incorporation of [U-¹⁴C]glucose into glyceride glycerol and fatty acids in normal fat cells in a manner dependent upon the glucose concentration. 2. In the presence of insulin the incorporation of 5mm-glucose into glyceride fatty acids was increased by concentrations of palmitate, adrenaline and 6-N-2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate up to 0.5mm, 0.5μm and 0.5mm respectively. Higher concentrations of these agents produced progressive decreases in the rate of glucose incorporation into fatty acids. 3. The effects of palmitate and lipolytic agents upon the measured parameters of glucose utilization were similar, suggesting that the effects of lipolytic agents are mediated through increased concentrations of free fatty acids. 4. In fat cells from 24h-starved rats, maximal stimulation of glucose incorporation into fatty acids was achieved with 0.25mm-palmitate. Higher concentrations of palmitate were inhibitory. In fat cells from 72h-starved rats, palmitate only stimulated glucose incorporation into fatty acids at high concentrations of palmitate (1mm and above). 5. The ability of fat cells to incorporate glucose into glyceride glycerol in the presence of palmitate decreased with increasing periods of starvation. 6. It is suggested that low concentrations of free fatty acids stimulate fatty acid synthesis from glucose by increasing the utilization of ATP and cytoplasmic NADH for esterification of these free fatty acids. When esterification of free fatty acids does not keep pace with their provision, inhibition of fatty acid synthesis occurs. Provision of free fatty acids far in excess of the esterification capacity of the cells leads to uncoupling of oxidative phosphorylation and a secondary stimulation of fatty acid synthesis from glucose.     

Physical and Kinetic Properties of the Nicotinamide Adenine Dinucleotide-specific Glutamate Dehydrogenase Purified from Chlorella sorokiniana

The nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (l-glutamate:NAD⁺ oxidoreductase, EC 1.4.1.2) of Chlorella sorokiniana was purified 1,000-fold to electrophoretic homogeneity. The native enzyme was shown to have a molecular weight of 180,000 and to be composed of four identical subunits with a molecular weight of 45,000. The N-terminal amino acid was determined to be lysine. The pH optima for the aminating and deaminating reactions were approximately 8 and 9, respectively. The K_m values for α-ketoglutarate, NADH, NH₄⁺, NAD⁺, and l-glutamate were 2 mm, 0.15 mm, 40 mm, 0.15 mm, and 60 mm, respectively. Whereas the K_m for α-ketoglutarate and l-glutamate increased 10-fold, 1 pH unit above or below the pH optima for the aminating or deaminating reactions, respectively, the K_m values for NADH and NAD⁺ were independent of change in pH from 7 to 9.6. By initial velocity, product inhibition, and equilibrium substrate exchange studies, the kinetic mechanism of enzyme was shown to be consistent with a bi uni uni uni ping-pong addition sequence. Although this kinetic mechanism differs from that reported for any other glutamate dehydrogenase, the chemical mechanism still appears to involve the formation of a Schiff base between α-ketoglutarate and an ε-amino group of a lysine residue in the enzyme. The physical, chemical, and kinetic properties of this enzyme differ greatly from those reported for the NH₄⁺-inducible glutamate dehydrogenase in this organism.     

Bacterial catabolism of threonine. Threonine degradation initiated by l-threonine acetaldehyde-lyase (aldolase) in species of Pseudomonas

1. The route of l-threonine degradation was studied in four strains of the genus Pseudomonas able to grow on the amino acid and selected because of their high l-threonine aldolase activity. Growth and manometric results were consistent with the cleavage of l-threonine to acetaldehyde+glycine and their metabolism via acetate and serine respectively. 2. l-Threonine aldolases in these bacteria exhibited pH optima in the range 8.0–8.7 and K_m values for the substrate of 5–10mm. Extracts exhibited comparable allo-l-threonine aldolase activities, K_m values for this substrate being 14.5–38.5mm depending on the bacterium. Both activities were essentially constitutive. Similar activity ratios in extracts, independent of growth conditions, suggested a single enzyme. The isolate Pseudomonas D2 (N.C.I.B. 11097) represents the best source of the enzyme known. 3. Extracts of all the l-threonine-grown pseudomonads also possessed a CoA-independent aldehyde dehydrogenase, the synthesis of which was induced, and a reversible alcohol dehydrogenase. The high acetaldehyde reductase activity of most extracts possibly resulted in the underestimation of acetaldehyde dehydrogenase. 4. l-Serine dehydratase formation was induced by growth on l-threonine or acetate+glycine. Constitutively synthesized l-serine hydroxymethyltransferase was detected in extracts of Pseudomonas strains D2 and F10. The enzyme could not be detected in strains A1 and N3, probably because of a highly active `formaldehyde-utilizing' system. 5. Ion-exchange and molecular exclusion chromatography supported other evidence that l-threonine aldolase and allo-l-threonine aldolase activities were catalysed by the same enzyme but that l-serine hydroxymethyltransferase was distinct and different. These results contrast with the specificities of some analogous enzymes of mammalian origin.     

Genetic control and biosynthesis of two new flavone-glycosides in the petals of Melandrium album

In a chemicogenetic analysis of the geographical distribution of flavone-glycosides in the petals of Melandrium album, we found two unknown flavone-glycosides in ten Hungarian and four German populations. By means of classical techniques for the identification and structure determination of flavonoids, the structure of these flavones turned out to be 6-C-glucosylglucosylapigenin and 7-O-glucosyl-6-C-glucosylglucosylapigenin, respectively. Genetic analysis showed that the coupling of glucose to the carbon-carbon bound glucose of isovitexin (6-C-glucosylapigenin) was controlled by a single dominant gene, Fg. Fg controls a UDP-glucose: isovitexin 6-C-glucosylglucosyltransferase. By means of ammonium sulfate fractionation and Sephadex chromatography, the enzyme was purified sixfold. The partly purified enzyme had a pH optimum between 8.0 and 8.5. The apparent K _m value for UDP-glucose in the presence of 1.0 mm isovitexin was 2.2 mm. The apparent K _m value for isovitexin in the presence of 1.8 mm UDP-glucose was 0.08 mm. The glucosyltransferase activity was stimulated by the divalent cations Mn, Mg, Co, and Ca. Neither 7-O-glucosyl nor 7-O-xylosyl isovitexin could serve as an enzyme substrate. Therefore, the biosynthesis of 7-O-glucosyl-6-C-glucosylglucosylapigenin found in the petals of M. album proceeds in a sequential manner: first the formation of 6-C-glucosylglucosylapigenin, followed by 7-O-glucosylation. Isovitexin 6-C-glucosylglucosyltransferase activity controlled by gene Fg could also be demonstrated in leaves of Fg plants. The enzyme probably uses another substrate in these green parts of the plant, because both isovitexin and isovitexin-glycosides are absent.     

Partial Purification and Properties of d-Glucosamine 6-Phosphate N-Acetyltransferase from Phaseolus aureus

d-Glucosamine-6-P N-acetyltransferase (EC 2.3.1.4) from mung bean seeds (Phaseolus aureus) was purified 313-fold by protamine sulfate and isoelectric precipitation, ammonium sulfate and acetone fractionation, and CM Sephadex column chromatography. The partially purified enzyme was highly specific for d-glucosamine-6-P. Neither d-glucosamine nor d-galactosamine could replace this substrate. The partially purified enzyme preparation was inhibited up to 50% by 2 × 10⁻²m EDTA, indicating the requirement of a divalent cation. Among divalent metal ions tested, Mg²⁺ was required for maximum activity of the enzyme. Mn²⁺ and Zn²⁺ were inhibitory, while Co²⁺ had no effect on the enzyme activity. The pH optimum of the enzyme in sodium acetate and sodium citrate buffers was found to be 5.2. The effect of Mg²⁺ on the enzyme in sodium acetate and sodium citrate buffers was particularly noticeable in the range of optimum pH. Km values of 15.1 × 10⁻⁴m and 7.1 × 10⁻⁴m were obtained for d-glucosamine-6-P and acetyl CoA, respectively. The enzyme was completely inhibited by 1 × 10⁻⁴mp-hydroxymercuribenzoate, and this inhibition was partially reversed by l-cysteine; indicating the presence of sulfhydryl groups at or near the active site of the enzyme.