Kinetics and mechanism of catalysis by proteolytic enzymes. A comparison of the kinetics of hydrolysis of synthetic substrates by bovine α- and β-trypsin

Several esters of the α-N-toluene-p-sulphonyl and α-N-benzoyl derivatives of S-(3-aminopropyl)-l-cysteine and the methyl ester of S-(4-aminobutyl)-N-toluene-p-sulphonyl-l-cysteine were synthesized. The kinetics of hydrolysis of these and esters of the α-N-toluene-p-sulphonyl and α-N-benzoyl derivatives of l-arginine, l-lysine, S-(2-aminoethyl)-l-cysteine and esters of γ-guanidino-l-α-toluene-p-sulphonamidobutyric acid and α-N-toluene-p-sulphonyl-l-homoarginine by α- and β-trypsin were compared. On the basis of values of the specificity constants (k_cat./K_m), the two enzymes display similar catalytic efficiency towards some substrates. In other cases α-trypsin is less efficient than β-trypsin. It is possible that α-trypsin possesses greater molecular flexibility than β-trypsin.     

alpha-l-Arabinofuranosidase from Radish (Raphanus sativus L.) Seeds

An α-l-arabinofuranosidase has been purified 1043-fold from radish (Raphanus sativus L.) seeds. The purified enzyme was a homogeneous glycoprotein consisting of a single polypeptide with an apparent molecular weight of 64,000 and an isoelectric point value of 4.7, as evidenced by denaturing gel electrophoresis and reversed-phase or size-exclusion high-performance liquid chromatography and isoelectric focusing. The enzyme characteristically catalyzes the hydrolysis of p-nitrophenyl α-l-arabinofuranoside and p-nitrophenyl β-d-xylopyranoside in a constant ratio (3:1) of the initial velocities at pH 4.5, whereas the corresponding α-l-arabinopyranoside and β-d-xylofuranoside are unsusceptible. The following evidence was provided to support that a single enzyme with one catalytic site was responsible for the specificity: (a) high purity of the enzyme preparation, (b) an invariable ratio of the activities toward the two substrates throughout the purification steps, (c) a parallelism of the activities in activation with bovine serum albumin and in heat inactivation of the enzyme as well as in the inhibition with heavy metal ions and sugars such as Hg²⁺, Ag⁺, l-arabino-(1→4)-lactone, and d-xylose, and (d) results of the mixed substrate kinetic analysis using the two substrates. The enzyme was shown to split off α-l-arabinofuranosyl residues in sugar beet arabinan, soybean arabinan-4-galactan, and radish seed and leaf arabinogalactan proteins. Arabinose and xylose were released by the action of the enzyme on oat-spelt xylan. Synergistic action of α-l-arabinofuranosidase and β-d-galactosidase on radish seed arabinogalactan protein resulted in the extensive degradation of the carbohydrate moiety.     

Purification and Substrate Specificities of Two α-l-Arabinofuranosidases from Aspergillus awamori IFO 4033

α-l-Arabinofuranosidases I and II were purified from the culture filtrate of Aspergillus awamori IFO 4033 and had molecular weights of 81,000 and 62,000 and pIs of 3.3 and 3.6, respectively. Both enzymes had an optimum pH of 4.0 and an optimum temperature of 60°C and exhibited stability at pH values from 3 to 7 and at temperatures up to 60°C. The enzymes released arabinose from p-nitrophenyl-α-l-arabinofuranoside, O-α-l-arabinofuranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose, and arabinose-containing polysaccharides but not from O-β-d-xylopyranosyl-(1→2)-O-α-l-arabinofuranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose. α-l-Arabinofuranosidase I also released arabinose from O-β-d-xylopy-ranosyl-(1→4)-[O-α-l-arabinofuranosyl-(1→3)]-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose. However, α-l-arabinofuranosidase II did not readily catalyze this hydrolysis reaction. α-l-Arabinofuranosidase I hydrolyzed all linkages that can occur between two α-l-arabinofuranosyl residues in the following order: (1→5) linkage > (1→3) linkage > (1→2) linkage. α-l-Arabinofuranosidase II hydrolyzed the linkages in the following order: (1→5) linkage > (1→2) linkage > (1→3) linkage. α-l-Arabinofuranosidase I preferentially hydrolyzed the (1→5) linkage of branched arabinotrisaccharide. On the other hand, α-l-arabinofuranosidase II preferentially hydrolyzed the (1→3) linkage in the same substrate. α-l-Arabinofuranosidase I released arabinose from the nonreducing terminus of arabinan, whereas α-l-arabinofuranosidase II preferentially hydrolyzed the arabinosyl side chain linkage of arabinan.Recently, it has been proven that l-arabinose selectively inhibits intestinal sucrase in a noncompetitive manner and reduces the glycemic response after sucrose ingestion in animals (33). Based on this observation, l-arabinose can be used as a physiologically functional sugar that inhibits sucrose digestion. Effective l-arabinose production is therefore important in the food industry. l-Arabinosyl residues are widely distributed in hemicelluloses, such as arabinan, arabinoxylan, gum arabic, and arabinogalactan, and the α-l-arabinofuranosidases (α-l-AFases) (EC 3.2.1.55) have proven to be essential tools for enzymatic degradation of hemicelluloses and structural studies of these compounds.α-l-AFases have been classified into two families of glycanases (families 51 and 54) on the basis of amino acid sequence similarities (11). The two families of α-l-AFases also differ in substrate specificity for arabinose-containing polysaccharides. Beldman et al. summarized the α-l-AFase classification based on substrate specificities (3). One group contains the Arafur A (family 51) enzymes, which exhibit very little or no activity with arabinose-containing polysaccharides. The other group contains the Arafur B (family 54) enzymes, which cleave arabinosyl side chains from polymers. However, this classification is too broad to define the substrate specificities of α-l-AFases. There have been many studies of the α-l-AFases (3, 12), especially the α-l-AFases of Aspergillus species (2–8, 12–15, 17, 22, 23, 28–32, 36–39, 41–43, 46). However, there have been only a few studies of the precise specificities of these α-l-AFases. In previous work, we elucidated the substrate specificities of α-l-AFases from Aspergillus niger 5-16 (17) and Bacillus subtilis 3-6 (16, 18), which should be classified in the Arafur A group and exhibit activity with arabinoxylooligosaccharides, synthetic methyl 2-O-, 3-O-, and 5-O-arabinofuranosyl-α-l-arabinofuranosides (arabinofuranobiosides) (20), and methyl 3,5-di-O-α-l-arabinofuranosyl-α-l-arabinofuranoside (arabinofuranotrioside) (19).In the present work, we purified two α-l-AFases from a culture filtrate of Aspergillus awamori IFO 4033 and determined the substrate specificities of these α-l-AFases by using arabinose-containing polysaccharides and the core oligosaccharides of arabinoxylan and arabinan.     

Elucidation of the Molecular Basis for Arabinoxylan-Debranching Activity of a Thermostable Family GH62 α-l-Arabinofuranosidase from Streptomyces thermoviolaceus

Xylan-debranching enzymes facilitate the complete hydrolysis of xylan and can be used to alter xylan chemistry. Here, the family GH62 α-l-arabinofuranosidase from Streptomyces thermoviolaceus (SthAbf62A) was shown to have a half-life of 60 min at 60°C and the ability to cleave α-1,3 l-arabinofuranose (l-Araf) from singly substituted xylopyranosyl (Xylp) backbone residues in wheat arabinoxylan; low levels of activity on arabinan as well as 4-nitrophenyl α-l-arabinofuranoside were also detected. After selective removal of α-1,3 l-Araf substituents from disubstituted Xylp residues present in wheat arabinoxylan, SthAbf62A could also cleave the remaining α-1,2 l-Araf substituents, confirming the ability of SthAbf62A to remove α-l-Araf residues that are (1→2) and (1→3) linked to monosubstituted β-d-Xylp sugars. Three-dimensional structures of SthAbf62A and its complex with xylotetraose and l-arabinose confirmed a five-bladed β-propeller fold and revealed a molecular Velcro in blade V between the β1 and β21 strands, a disulfide bond between Cys27 and Cys297, and a calcium ion coordinated in the central channel of the fold. The enzyme-arabinose complex structure further revealed a narrow and seemingly rigid l-arabinose binding pocket situated at the center of one side of the β propeller, which stabilized the arabinofuranosyl substituent through several hydrogen-bonding and hydrophobic interactions. The predicted catalytic amino acids were oriented toward this binding pocket, and the catalytic essentiality of Asp53 and Glu213 was confirmed by site-specific mutagenesis. Complex structures with xylotetraose revealed a shallow cleft for xylan backbone binding that is open at both ends and comprises multiple binding subsites above and flanking the l-arabinose binding pocket.     

Inhibition of glycosidases by aldonolactones of corresponding configuration. The specificity of α-l-arabinosidase

1. The previous study (Conchie, Gelman & Levvy, 1967b) of the specificity of β-glucosidase, β-galactosidase and β-d-fucosidase in barley, limpet, almond emulsin and rat epididymis was extended to α-l-arabinosidase. 2. The inhibitory action of l-arabinono-(1→5)-lactone was tested against all four types of enzyme, and α-l-arabinosidase was examined for inhibition by glucono-, galactono- and d-fucono-lactone. 3. In emulsin, the enzyme that hydrolyses β-glucosides, β-galactosides and β-d-fucosides also hydrolyses α-l-arabinosides. Rat epididymis resembles emulsin except that, as already noted, it lacks β-glucosidase activity. 4. In the limpet, α-l-arabinosidase activity is associated with the enzyme that hydrolyses β-glucosides and β-d-fucosides, and not with the separate β-galactosidase. 5. The effects of the different lactones on the barley preparation suggest that α-l-arabinosidase activity is associated with the β-galactosidase rather than with the enzyme that hydrolyses β-glucosides and β-d-fucosides. Fractionation and heat-inactivation experiments indicate that there is also a separate α-l-arabinosidase in the preparation.     

A beta-Galactosidase from Radish (Raphanus sativus L.) Seeds

A basic β-galactosidase (β-Galase) has been purified 281-fold from imbibed radish (Raphanus sativus L.) seeds by conventional purification procedures. The purified enzyme is an electrophoretically homogeneous protein consisting of a single polypeptide with an apparent molecular mass of 45 kilodaltons and pl values of 8.6 to 8.8. The enzyme was maximally active at pH 4.0 on p-nitrophenyl β-d-galactoside and β-1,3-linked galactobiose. The enzyme activity was inhibited strongly by Hg²⁺ and 4-chloromercuribenzoate. d-Galactono-(1→4)-lactone and d-galactal acted as potent competitive inhibitors. Using galactooligosaccharides differing in the types of linkage as the substrates, it was demonstrated that radish seed β-Galase specifically split off β-1,3- and β-1,6-linked d-galactosyl residues from the nonreducing ends, and their rates of hydrolysis increased with increasing chain lengths. Radish seed and leaf arabino-3,6-galactan-proteins were resistant to the β-galase alone but could be partially degraded by the enzyme after the treatment with a fungal α-l-arabinofuranosidase leaving some oligosaccharides consisting of d-galactose, uronic acid, l-arabinose, and other minor sugar components besides d-galactose as the main product.     

Proteases and Peptidases of Castor Bean Endosperm: Enzyme Characterization and Changes during Germination

The endosperm of castor bean seeds (Ricinus communis L.) contains two —SH-dependent aminopeptidases, one hydrolyzing l-leucine-β-naphthylamide optimally at pH 7.0, and the other hydrolyzing l-proline-β-naphthylamide optimally at pH 7.5. After germination the endosperm contains in addition an —SH-dependent hemoglobin protease, a serine-dependent carboxypeptidase, and at least two —SH-dependent enzymes hydrolyzing the model substrate α-N-benzoyl-dl-arginine-β-naphthylamide (BANA). The carboxypeptidase is active on a variety of N-carbobenzoxy dipeptides, especially N-carbobenzoxy-L-phenylalanine-l-alanine and N-carbobenzoxy-l-tyrosine-l-leucine. The pH optima for the protease, carboxypeptidase, and BANAase acivities are 3.5 to 4.0, 5.0 to 5.5, and 6 to 8, respectively.     

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.     

β-Galactosidase-catalysed hydrolysis of β-d-galactopyranosyl azide

1. β-d-Galactopyranosyl azide is hydrolysed by the β-galactosidase of Escherichia coli to galactose and azide ion at a mechanistically significant rate. 2. Methyl 1-thio-β-d-galactopyranoside is a competitive inhibitor of the hydrolysis of the azide and of o-nitrophenyl β-d-galactopyranoside with K_i 1.8mm. 3. β-Galactosidase can thus hydrolyse a range of substrates of general structure β-d-galactopyranosyl-X(Y), where the atom X has a lone pair of electrons on which the enzyme may act as a Lewis or Brønsted acid, but in which the length of the bond cleaved varies significantly, which is inconsistent with the orbital steering hypothesis.     

Properties of an Aminotransferase of Pea (Pisum sativum L.)

A transaminase (aminotransferase, EC 2.6.1) fraction was partially purified from shoot tips of pea (Pisum sativum L. cv. Alaska) seedlings. With α-ketoglutarate as co-substrate, the enzyme transaminated the following aromatic amino acids: d,l-tryptophan, d,l-tyrosine, and d,l-phenylalanine, as well as the following aliphatic amino acids: d,l-alanine, d,l-methionine, and d,l-leucine. Of other α-keto acids tested, pyruvate and oxalacetate were more active than α-ketoglutarate with d,l-tryptophan. Stoichiometric yields of indolepyruvate and glutamate were obtained with d,l-tryptophan and α-ketoglutarate as co-substrates. The specific activity was three times higher with d-tryptophan than with l-tryptophan.     

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.     

The pectic disaccharides lepidimoic acid and β-d-xylopyranosyl-(1→3)-d-galacturonic acid occur in cress-seed exudate but lack allelochemical activity

Background and aims Cress-seed (Lepidium sativum) exudate exerts an allelochemical effect, promoting excessive hypocotyl elongation and inhibiting root growth in neighbouring Amaranthus caudatus seedlings. We investigated acidic disaccharides present in cress-seed exudate, testing the proposal that the allelochemical is an oligosaccharin—lepidimoic acid (LMA; 4-deoxy-β-l-threo-hex-4-enopyranuronosyl-(1→2)-l-rhamnose).Methods Cress-seed exudate was variously treated [heating, ethanolic precipitation, solvent partitioning, high-voltage paper electrophoresis and gel-permeation chromatography (GPC)], and the products were bioassayed for effects on dark-grown Amaranthus seedlings. Two acidic disaccharides, including LMA, were isolated and characterized by electrophoresis, thin-layer chromatography (TLC) and nuclear magnetic resonance (NMR) spectroscopy, and then bioassayed.Key Results Cress-seed exudate contained low-M_r, hydrophilic, heat-stable material that strongly promoted Amaranthus hypocotyl elongation and inhibited root growth, but that separated from LMA on electrophoresis and GPC. Cress-seed exudate contained ∼250 µm LMA, whose TLC and electrophoretic mobilities, susceptibility to mild acid hydrolysis and NMR spectra are reported. A second acidic disaccharide, present at ∼120 µm, was similarly characterized, and shown to be β-d-xylopyranosyl-(1→3)-d-galacturonic acid (Xyl→GalA), a repeat unit of xylogalacturonan. Purified LMA and Xyl→GalA when applied at 360 and 740 µm, respectively, only slightly promoted Amaranthus hypocotyl growth, but equally promoted root growth and thus had no effect on the hypocotyl:root ratio, unlike total cress-seed exudate.Conclusions LMA is present in cress seeds, probably formed by rhamnogalacturonan lyase action on rhamnogalacturonan-I during seed development. Our results contradict the hypothesis that LMA is a cress allelochemical that appreciably perturbs the growth of potentially competing seedlings. Since LMA and Xyl→GalA slightly promoted both hypocotyl and root elongation, their effect could be nutritional. We conclude that rhamnogalacturonan-I and xylogalacturonan (pectin domains) are not sources of oligosaccharins with allelochemical activity, and the biological roles (if any) of the disaccharides derived from them are unknown. The main allelochemical principle in cress-seed exudate remains to be identified.     

Identification and Quantitative Analysis of Indole-3-Acetyl-l-Aspartate from Seeds of Glycine max L

Indole-3-acetyl-l-aspartate (IAAsp) was isolated from seeds of Glycine max L. cv. Hark and its identity established by its chromatographic performance and its mass spectral fragmentation. Following acid hydrolysis, the aspartate moiety was shown to be the l-enantiomer by reverse phase high performance liquid chromatographic retention time of the bisethyl ester derivatized with 2,3,4,6-tetra-O-acetyl-β-d-glycopyranosyl isothiocyanate. Isotope dilution analysis using [¹⁴C]IAAsp as internal standard showed that soybean seed contained 10 μmol/kg IAAsp and this accounted for one-half of the total indoleacetic acid of the seed.     

Sugar-nucleotide precursors of arabinopyranosyl, arabinofuranosyl, and xylopyranosyl residues in spinach polysaccharides

Cultured spinach (Spinacia oleracea L. cv Monstrous Viroflay) cells incorporated exogenous l-[³H]arabinose sequentially into β-l-arabinopyranose-1-phosphate, uridine diphospho-β-l-arabinopyranose, uridine diphospho-α-d-xylopyranose and (in some experiments) α-d-xylopyranose-1-phosphate. The amount of ³H in each of these compounds reached a plateau after a few minutes, and could be rapidly chased with nonradioactive l-arabinose, demonstrating rapid turnover. After a few minutes' lag, incorporation of ³H into the arabinofuranosyl, arabinopyranosyl, and xylopyranosyl residues of polysaccharides was linear with respect to time. The kinetics of labeling were compatible with UDP-β-l-arabinopyranose and UDP-α-d-xylopyranose being the immediate precursors of arabians (both the pyranose and the furanose residues) and xylans, respectively. No other radioactive nucleotides were formed; in particular, UDP-arabinofuranose was absent. There was no evidence for conversion of arabinopyranose to arabinofuranose within the polysaccharides, suggesting that this conversion occurs during polymer synthesis. The glycolipids detected showed too slow a turnover to be intermediates of pentosan synthesis.     

Identification and Characterization of a Novel Terrabacter ginsenosidimutans sp. nov. β-Glucosidase That Transforms Ginsenoside Rb1 into the Rare Gypenosides XVII and LXXV

A new β-glucosidase from a novel strain of Terrabacter ginsenosidimutans (Gsoil 3082^T) obtained from the soil of a ginseng farm was characterized, and the gene, bgpA (1,947 bp), was cloned in Escherichia coli. The enzyme catalyzed the conversion of ginsenoside Rb1 {3-O-[β-d-glucopyranosyl-(1-2)-β-d-glucopyranosyl]-20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to the more pharmacologically active rare ginsenosides gypenoside XVII {3-O-β-d-glucopyranosyl-20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol}, gypenoside LXXV {20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol}, and C-K [20-O-(β-d-glucopyranosyl)-20(S)-protopanaxadiol]. A BLAST search of the bgpA sequence revealed significant homology to family 3 glycoside hydrolases. Expressed in E. coli, β-glucosidase had apparent K_m values of 4.2 ± 0.8 and 0.14 ± 0.05 mM and V_max values of 100.6 ± 17.1 and 329 ± 31 μmol·min⁻¹·mg of protein⁻¹ against p-nitrophenyl-β-d-glucopyranoside and Rb1, respectively. The enzyme catalyzed the hydrolysis of the two glucose moieties attached to the C-3 position of ginsenoside Rb1, and the outer glucose attached to the C-20 position at pH 7.0 and 37°C. These cleavages occurred in a defined order, with the outer glucose of C-3 cleaved first, followed by the inner glucose of C-3, and finally the outer glucose of C-20. These results indicated that BgpA selectively and sequentially converts ginsenoside Rb1 to the rare ginsenosides gypenoside XVII, gypenoside LXXV, and then C-K. Herein is the first report of the cloning and characterization of a novel ginsenoside-transforming β-glucosidase of the glycoside hydrolase family 3.Ginseng refers to the roots of members of the plant genus Panax, which have been used as a traditional medicine in Asian countries for over 2,000 years due to their observed beneficial effects on human health. Ginseng saponins, also referred to as ginsenosides, are the major active components of ginseng (27). Various biological activities have been ascribed to ginseng saponins, including anti-inflammatory activity (43), antitumor effects (23, 39), and neuroprotective and immunoprotective (15, 31) effects.Ginsenosides can be categorized as protopanaxadiol (PPD), protopanaxatriol, and oleanane saponins, based on the structure of the aglycon, with a dammarane skeleton (29). The PPD-type ginsenosides are further classified into subgroups based on the position and number of sugar moieties attached to the aglycon at positions C-3 and C-20. For example, one of the largest PPD-type ginsenosides, Rb1 {3-O-[β-d-glucopyranosyl-(1-2)-β-d-glucopyranosyl]-20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol}, contains 4 glucose moieties, two each attached via glycosidic linkages to the C-3 and C-20 positions of the aglycon (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Chemical structures of protopanaxadiol and protopanaxatriol ginsenosides (5). The ginsenosides represented here are all (S)-type ginsenosides. glc, β-d-glucopyranosyl; arap, α-l-arabinopyranosyl; araf, α-l-arabinofuranosyl; rha, α-l-rhamnopyranosyl; Gyp, gypenoside; C, compound.Because of their size, low solubility, and poor permeability across the cell membrane, it is difficult for human body to directly absorb large ginsenosides (44), although these components constitute the major portion of the total ginsenoside in raw ginseng (30). Moreover, the lack of the availability of the rare ginsensoides limits the research on their biological and medicinal properties. Therefore, transformation of these major ginsenosides into smaller deglycosylated ginsenosides, which are more effective in in vivo physiological action, is required (1, 37).The production of large amounts of rare ginsenosides from the major ginsenosides can be accomplished through a number of physiochemical methods such as heating (17), acid treatment (2), and alkali treatment (48). However, these approaches produce nonspecific racemic mixtures of rare ginsenosides. As an alternative, enzymatic methods have been explored as a way to convert the major ginsenosides into more pharmacologically active rare ginsenosides in a more specific manner (14, 20).To date, three types of glycoside hydrolases, β-d-glucosidase, α-l-arabinopyranosidase, and α-l-arabinofuranosidase, have been found to be involved in the biotransformation of PPD-type ginsenosides. For example, a β-glucosidase isolated from a fungus converts Rb1 to C-K [20-O-(β-d-glucopyranosyl)-20(S)-protopanaxadiol] (45), and an α-l-arabinopyranosidase and α-l-arabinofuranosidase have been isolated from an intestinal bacterium that hydrolyze, respectively, Rb2 {3-O-[β-d-glucopyranosyl-(1-2)-β-d-glucopyranosyl]-20-O-[α-l-arabinopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to Rd {3-O-[β-d-glucopyranosyl-(1-2)-β-d-glucopyranosyl]-20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol} and Rc {3-O-[β-d-glucopyranosyl-(1-2)-β-d-glucopyranosyl]-20-O- [α-l-arabinofuranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to Rd (34). Two recombinant enzymes that convert major ginsenosides into rare ginsenosides have been cloned and expressed in Escherichia coli: Solfolobus solfataricus β-glycosidase, which transforms Rb1 or Rc to C-K (28), and β-glucosidase from a soil metagenome, which transforms Rb1 to Rd (16). Both of these glycoside hydrolases are family 1 glycoside hydrolases.Here, we report the cloning and expression in E. coli of a gene (bgpA) encoding a new ginsenoside-hydrolyzing β-glucosidase from a novel bacterial strain, Terrabacter ginsenosidimutans sp. nov. Gsoil 3082, isolated from a ginseng farm in Korea. BgpA is a family 3 glycoside hydrolase, and the recombinant enzyme employs a different enzymatic pathway from ginsenoside-hydrolyzing family 1 glycoside hydrolases. BgpA preferentially and sequentially hydrolyzed the terminal and inner glucoses at the C-3 position of ginsenoside Rb1 and then the outer glucose at the C-20 position. Thus, BgpA could be effective in the biotransformation of ginsenoside Rb1 to gypenoside (Gyp) XVII {3-O-β-d-glucopyranosyl-20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol}, Gyp LXXV {20-O-[β-d-glucopyranosyl-(1-6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol}, and C-K.     

Kinetics and mechanism of catalysis by proteolytic enzymes: The kinetics of hydrolysis of derivatives of l-lysine and S-(beta-aminoethyl)-l-cysteine(thialysine)by bovine trypsin

1. Several esters of the α-N-toluene-p-sulphonyl and N-benzoyl derivatives of l-lysine and S-(β-aminoethyl)-l-cysteine have been synthesized. 2. The kinetics of hydrolysis of the esters by bovine trypsin have been compared. Values of k₀ are similar for corresponding derivatives of the isosteric amino acids and deacylation of an acyl-enzyme appears to be rate-determining in each case. There are, however, some quantitative kinetic differences between the various series of substrates.     

A high proportion of hybridomas raised to a plant extract secrete antibody to arabinose or galactose

A high proportion of hybridomas, obtained from mice immunized with style extracts prepared from mature flowers of an ornamental tobacco, Nicotiana alata, secrete antibody to arabinogalactan protein (AGP). The specificity of the antibodies secreted by three cloned cell lines is primarily directed to β-d-galactopyranose and α-l-arabinofuranose; antibodies from two cell lines preferentially bind β-d-galactopyranose residues and antibodies from the other cell line preferentially bind α-l-arabinofuranose. As AGPs are components of most plant tissues and exudates, it is likely that attempts to raise monoclonal antibodies to other plant extracts will result in hybridomas producing antibodies to AGPs.     

Identification and Characterization of a Novel Secreted Glycosidase with Multiple Glycosidase Activities in Streptococcus intermedius

Streptococcus intermedius is a known human pathogen and belongs to the anginosus group (S. anginosus, S. intermedius, and S. constellatus) of streptococci (AGS). We found a large open reading frame (6,708 bp) in the lac operon, and bioinformatic analysis suggested that this gene encodes a novel glycosidase that can exhibit β-d-galactosidase and N-acetyl-β-d-hexosaminidase activities. We, therefore, named this protein “multisubstrate glycosidase A” (MsgA). To test whether MsgA has these glycosidase activities, the msgA gene was disrupted in S. intermedius. The msgA-deficient mutant no longer showed cell- and supernatant-associated β-d-galactosidase, β-d-fucosidase, N-acetyl-β-d-glucosaminidase, and N-acetyl-β-d-galactosaminidase activities, and all phenotypes were complemented in trans with a recombinant plasmid carrying msgA. Purified MsgA had all four of these glycosidase activities and exhibited the lowest K_m with 4-methylumbelliferyl-linked N-acetyl-β-d-glucosaminide and the highest k_cat with 4-methylumbelliferyl-linked β-d-galactopyranoside. In addition, the purified LacZ domain of MsgA had β-d-galactosidase and β-d-fucosidase activities, and the GH20 domain exhibited both N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities. The β-d-galactosidase and β-d-fucosidase activities of MsgA are thermolabile, and the optimal temperature of the reaction was 40°C, whereas almost all enzymatic activities disappeared at 49°C. The optimal temperatures for the N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities were 58 and 55°C, respectively. The requirement of sialidase treatment to remove sialic acid residues of the glycan branch end for glycan degradation by MsgA on human α₁-antitrypsin indicates that MsgA has exoglycosidase activities. MsgA and sialidase might have an important function in the production and utilization of monosaccharides from oligosaccharides, such as glycans for survival in a normal habitat and for pathogenicity of S. intermedius.     

Arabinogalactan-Proteins from Primary and Mature Roots of Radish (Raphanus sativus L.)

Organ-specific variations in blood group H-like activity were observed in developing radish plants. A temporary increase in serological activity was found to occur in the roots at the earlier stages of development. Arabinogalactan-proteins (AGPs) were isolated from primary and mature roots, and investigated for changes in their physicochemical properties, structure, and serological activities. These root AGPs were composed mainly of l-arabinose and d-galactose but were distinguishable from each other in their contents of l-fucose as well as of protein and hydroxyproline. The structures of the carbohydrate moieties of the root AGPs were essentially similar to those of AGPs isolated from seeds and mature leaves in that they consisted of consecutive (1→3)-linked β-d-galactosyl backbone chains having side chains of (1→6)-linked β-d-galactosyl residues, to which α-l-arabinofuranosyl residues were attached in the outer regions. One prominent feature of the primary root AGPs was that they contained appreciable amounts of l-fucose, which was presumably responsible for expression of the serological activity. In their immunological reactions with rabbit anti-radish leaf AGP antibody, the root AGPs were shown to share common antigenic determinant(s) with those of seed and leaf AGPs.     

Pre-steady-state Kinetic and Structural Analysis of Interaction of Methionine γ-Lyase from Citrobacter freundii with Inhibitors

Methionine γ-lyase (MGL) catalyzes the γ-elimination of l-methionine and its derivatives as well as the β-elimination of l-cysteine and its analogs. These reactions yield α-keto acids and thiols. The mechanism of chemical conversion of amino acids includes numerous reaction intermediates. The detailed analysis of MGL interaction with glycine, l-alanine, l-norvaline, and l-cycloserine was performed by pre-steady-state stopped-flow kinetics. The structure of side chains of the amino acids is important both for their binding with enzyme and for the stability of the external aldimine and ketimine intermediates. X-ray structure of the MGL·l-cycloserine complex has been solved at 1.6 Å resolution. The structure models the ketimine intermediate of physiological reaction. The results elucidate the mechanisms of the intermediate interconversion at the stages of external aldimine and ketimine formation.