Transglucosylation of tertiary alcohols using cassava beta-glucosidase

We have compared the ability of beta-glucosidases from cassava, Thai rosewood, and almond to synthesize alkyl glucosides by transglucosylating alkyl alcohols of chain length C(1)-C(8). Cassava linamarase shows greater ability to transfer glucose from p-nitrophenyl-beta-glucoside to secondary alcohol acceptors than other beta-glucosidases, and is unique in being able to synthesize C(4), C(5), and C(6) tertiary alkyl beta-glucosides with high yields of 94%, 82%, and 56%, respectively. Yields of alkyl glucosides could be optimized by selecting appropriate enzyme concentrations and incubation times. Cassava linamarase required pNP-glycosides as donors and could not use mono- or di-saccharides as sugar donors in alkyl glucoside synthesis.     

Kinetic analysis of the mechanism of action of beta-glucosidase from Botryodiplodia theobromae Pat.

1. The kinetic mechanism of beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) of Botryodiplodia theobromae Pat. has been studied in the presence of competing glucosyl acceptors. 2. Glycerol, fructose, sucrose, cellobiose and to a much lesser extent, maltose can act as glucosyl acceptors, apart from water. 3. Evidence confirming and supporting the kinetic mechanism previously postulated (Umezurike, G.M. (1971) Biochim. Biophys. Acta. 250, 182-191) is presented. 4. A theoretical kinetic analysis of the behaviour of the enzyme in the presence of two alternative glucosyl acceptors in addition to water is found to be consistent with experimental observation, suggesting a system in which both donor and acceptors bind to the enzyme in a random fashion to form ternary complexes. 5. The results are discussed in terms of the mechanism of group-transfer reactions.     

Galactosylsphingosine inhibition of the broad-specificity cytosolic beta-glucosidase of human liver

Glucosylsphingosine is a potent inhibitor of lysosomal glucocerebrosidase and the broad-specificity, cytosolic beta-glucosidase of human liver. In the present study, it was demonstrated that the broad-specificity beta-glucosidase was also inhibited by galactosylsphingosine. The inhibition was observed when the enzyme was assayed for beta-glucosidase, beta-galactosidase, beta-xylosidase, and alpha-arabinosidase activities. Inhibition was of the mixed-type and the degree of inhibition depended on the substrate. For example, galactosylsphingosine was a more potent inhibitor of beta-glucosidase activity (I0.5 = 0.3 mM) than beta-xylosidase activity (I0.5 = 1.2 mM). In addition, the observation that the broad-specificity, cytosolic beta-glucosidase was inhibited by hydrophobic glycosphingolipids prompted the definition of a revised purification procedure which took advantage of hydrophobic affinity chromatography. This revised purification scheme employed Octyl-Sepharose and yielded the largest (68,000 Da) and most active (470,000 nmol h-1 mg protein-1) beta-glucosidase preparation yet described. The beta-glucosidase preparation contained 19% serine and 17% glycine, while 24% of the total amino acids were hydrophobic. The results of this study document the presence of a sphingolipid binding site on the broad-specificity beta-glucosidase. The implications of galactosylsphingosine inhibition of cytosolic beta-glucosidase and the possible role of the enzyme in glycosphingolipid metabolism are discussed.     

Crystal structure of the covalent intermediate of human cytosolic beta-glucosidase

Human cytosolic β-glucosidase, also known as klotho-related protein (KLrP, GBA3), is an enzyme that hydrolyzes various β-d-glucosides, including glucosylceramide. We recently reported the crystal structure of KLrP in complex with glucose [Y. Hayashi, N. Okino, Y. Kakuta, T. Shikanai, M. Tani, H. Narimatsu, M. Ito, Klotho-related protein is a novel cytosolic neutral beta-glycosylceramidase, J. Biol. Chem. 282 (2007) 30889-30900]. Here, we report the crystal structure of a covalent intermediate of the KLrP mutant E165Q, in which glucose was covalently bound to a nucleophile, Glu³⁷³. The structure confirms the double displacement mechanism of the retaining β-glucosidase. In addition, the structure suggests that a water molecule could be involved in the stabilization of transition states through a sugar, 2-hydroxyl.     

Deciphering enzymes. Genetic selection as a probe of structure and mechanism.

The efficient engineering of enzymes with novel activities remains an ongoing challenge. Towards this end, genetic selection techniques provide a method for finding rare solutions to catalytic problems that requires only a limited foreknowledge of structure-function relationships. We have used genetic selections to extensively probe the structure and mechanism of chorismate mutases. The insights gained from these investigations will aid future enzyme design efforts.     

Kinetic properties and mechanism of action of an intracellular beta-glucosidase from Thermoascus aurantiacus Miehe

Intracellular beta-glucosidase is strongly inhibited by its own substrate p-nitrophenyl-beta-glucoside which displays high affinity for two binding sites. A non-productive complex is formed also by cellobiose, but its lower affinity results in a much lower inhibition. As shown by inhibition experiments performed with glucono-delta-lactone, the hydrolytic reaction proceeds through the formation of a carbonium ion, very similar in its half-chair conformation to the delta-lactone. Carboxylic groups (pK = 3.19) appear involved in the catalytic process together with a histidine residue (pK = 5.64): while the carboxylate ions stabilize the carbonium ion, the displaced group accepts a proton from the protonated imidazole.     

Exolytic hydrolysis of toxic plant glucosides by guinea pig liver cytosolic beta-glucosidase.

We demonstrate that although the guinea pig liver cytosolic beta-glucosidase does not catalyze the hydrolysis of gentiobiose, it does hydrolyze, disaccharide-containing glycosides such as p-nitrophenyl-beta-D-gentiobioside (Glc beta 1----6Glc beta-pNP) and mandelonitrile-beta-D-gentiobioside (amygdalin). Furthermore, we establish that the enzyme attacks disaccharide glycosides exolytically; specifically, we document the exolytic deglucosylation of amygdalin and the generation of the intermediate monosaccharide glycoside mandelonitrile-beta-D-glucoside prior to the formation of the aglycone (mandelonitrile). We also show that the cytosolic beta-glucosidase catalyzes the hydrolysis of various phenolic (e.g. arbutin and salicin) and cyanogenic plant glucosides (e.g. prunasin). Using the everted gut-sack technique, we demonstrate that the plant glucosides, amygdalin, prunasin, and vicine, are transported across the small intestine of the guinea pig efficiently and without being hydrolyzed. Based on these data we speculate that the cytosolic beta-glucosidase may participate in biotransformation of toxic plant glucosides.     

Isotope exchange as a probe of the kinetic mechanism of pyrophosphate-dependent phosphofructokinase

Data obtained from isotope exchange at equilibrium, exchange of inorganic phosphate against forward reaction flux, and positional isotope exchange of 18O from the bridge position of pyrophosphate to a nonbridge position all indicate that the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii has a rapid equilibrium random kinetic mechanism. The maximum rates of isotope exchange at equilibrium for the [14C]fructose 1,6-bisphosphate in equilibrium fructose 6-phosphate, [32P]Pi in equilibrium MgPPi, and Mg[32P]PPi in equilibrium fructose 1,6-bisphosphate exchange reactions increasing all four possible substrate-product pairs in constant ratio are identical, consistent with a rapid equilibrium mechanism. All exchange reactions are strongly inhibited at high concentrations of the fructose 6-phosphate (F6P)/Pi and MgPPi/Pi substrate-product pairs and weakly inhibited at high concentrations of the MgPPi/fructose 1,6-bisphosphate (FBP) pair suggesting three dead-end complexes, E:F6P:Pi, E:MgPPi:Pi, and E:FBP:MgPPi, in agreement with initial velocity studies [Bertagnolli, B.L., & Cook, P.F. (1984) Biochemistry 23, 4101]. Neither back-exchange by [32P]Pi nor positional isotope exchange of 18O-bridge-labeled pyrophosphate was observed under any conditions, suggesting that either the chemical interconversion step or a step prior to it limits the overall rate of the reaction.     

The beta-glucosidase from Botryodiplodia theobromae. Mechanism of enzyme action.

1. In the presence of a high concentration of p-nitrophenyl beta-D-glucopyranoside (donor) the rates of production of p-nitrophenol and a transglucosylation product (1-glyceryl beta-D-glucopyranoside) increased, whereas the rate of production of glucose decreased with increasing concentration of glycerol in reactions catalysed by the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) obtained from culture filtrates of Botryodiplodia theobromae Pat. 2. When [donor] greater than Km the rate of production of p-nitrophenol was higher in the presence of glycerol than in its absence, whereas when [donor] less than Km the rate of production of p-nitrophenol was lower in the presence of glycerol than in its absence. 3. Glycerol increased both the Michaelis constant (Km) and maximum velocity (Vmax.), whereas dioxan increased Km but decreased Vmax. 4. Up to 1 mM-AgNO3 had no effect on enzyme activity. 5. A 2H-solvent-isotope-effect [Vmax. (H2O)/V max. (2H2O)] value of 1.40 +/- 0.05 was found at pH (or p2H) 5.8 6. alpha-2H-kinetic isotope-effect (kappa H/kappa 2H) values of 1.03 +/- 0.01 and 1.05 +/- 0.01 were found in the absence and presence of glycerol respectively. 7. Although maltose was a non-competitive inhibitor of beta-glucosidase activity, the ratio of velocity in the presence of glycerol to that in its absence increased, after an initial decline, with increasing concentration of maltose. 8. These results are discussed in terms of a mechanism involving a solvent-separated glucosyl cation-carboxylate ion-pair, which has greater affinity for alcoholic glucosyl acceptors, and an intimate ion-pair, which has greater affinity for water as a glucosyl acceptor and which could collapse reversibly and rapidly into a preponderance of an unreactive covalent glucosyl-enzyme.     

Recombinant aequorin as a probe for cytosolic free Ca2+ in Escherichia coli

We describe a novel and simple method for the measurement of bacterial cytosolic free calcium ([Ca2+]i) using recombinant aequorin reconstituted within live bacterial cells. Using this method we have measured the effects of external calcium, complement, phagocytosis and antibiotics on the [Ca2+]i of Escherichia coli. In principle this method should be applicable to any genetically transformable organism and should suffer fewer problems than fluorescent dyes for subcellular calcium measurement.     

Energy of binding of Aspergillus oryzae beta-glucosidase with the substrate, and the mechanism of its enzymic action

Several beta-D-glucopyranosides (p-nitrophenyl, phenyl, and ethyl), 1-thio-beta-D-glucopyranosides, and phenyl 2-deoxy, 3-deoxy, 4-deoxy, and 6-deoxy beta-D-glucopyranosides were synthesized and used to study the mechanism of the enzymatic action of Taka-beta-glucosidase [EC 3.2.1.21 Aspergillus oryzae]. Kinetic constants of the enzyme for these glycosides were determined from S/V-S or 1/V-1/S plots, and the hydrolysis rates of these compounds with the enzyme, acid (3 N HCl) and alkali (3 N NaOH) were compared. Inhibition of the enzyme by 1,5-anhydroglucitol, glucal, dihydroglucal, and 1,6-anhydroglucopyranose was also examined. Glucal and 1,5-anhydroglucitol showed strong competitive inhibition. Free energy of binding of each hydroxyl group of glucosidic glucose with the enzyme was estimated from Kms of phenyl beta-glucoside and its deoxy analogues, and also Ki values of some inhibitors. The free energies of binding of 2-OH, 3-OH, 4-OH, and 6-OH were calculated to be 1.1, 2.4, 0.7, and 1.8 kcal/mol, respectively. The free energy of binding of phenoxide at C-1 (0.3 kcal/mol) was calculated from the Km of Ph-beta-Glc and Ki of 1,5-anhydroglucitol. The energy of binding of 5-CH2OH (2.3 kcal/mol) was obtained from the Km of Ph-beta-Glc and that of Ph-beta-Xyl. The sum (6.8 kcal/mol) of each partial binding free energy was close to the value of binding free energy of Ph-beta-Glc (7.0 kcal/mol) calculated by the equation; -delta Gbind = -RT ln Km-T delta Smix, showing that the methods of estimation of each binding energy used in the present study seemed reasonable. Glucal, having a pyranose form distorted slightly, showed strong competitive inhibition and the Ki of this inhibitor was smaller than the Km of Ph-beta-Glc, suggesting that the sugar ring bound to the active site was distored to a half chair form which is labile to acid hydrolysis.     

Dopamine inhibits cytosolic Ca2+ increases in rat lactotroph cells. Evidence of a dual mechanism of action

Single rat lactotroph cells were studied after loading with the cytosolic free Ca2+ concentration ([Ca2+]i) indicator fura-2 either 1 or 3 days after cell dispersion. Under unstimulated conditions, two groups of lactotrophs were observed, the first (predominant at day 1) with large [Ca2+]i fluctuations (peaks up to 300 nM) probably due to spontaneous action potentials and the second (predominant at 3 days) with stable [Ca2+]i (values variable between 65 and 200 nM). The effect of dopamine on the resting [Ca2+]i was different in the two groups. Even at high dopamine concentrations, no change occurred in the second group; whereas in the first, disappearance of fluctuations and marked decrease of [Ca2+]i were observed. These effects of dopamine appear to be due to hyperpolarization that was demonstrated by the use of a specific fluorescent indicator, bis(oxonol). Two types of triggered [Ca2+]i transients were studied in detail: those due to redistribution of Ca2+ from the intracellular stores (induced by thyrotropin-releasing hormone) and those due to Ca2+ influx through voltage-gated Ca2+ channels (induced by high [K+]). Dopamine (1 microM) markedly inhibited both these transients by the action of D2 receptors (blocked by 1-sulpiride and domperidone). All effects of dopamine were prevented by treatment of the cells with pertussis toxin, indicating the involvement of one (or more) GTP-binding protein(s). Another consequence of D2 receptor activation is the inhibition of adenylate cyclase. Treatments (cholera toxin, forskolin), known to raise cAMP levels, were found to dissociate the effects of dopamine on [Ca2+]i inasmuch as they markedly relieved the inhibition of the redistributive transients by thyrotropin-releasing hormone but left hyperpolarization and inhibition of K+ transients unaffected. The spectrum of intracellular signals elicited by the activation of D2 receptors is therefore complex and includes at least two mechanisms that involve [Ca2+]i, one related and the other independent of the decrease of cAMP levels.     

Thionesters as a probe for electrophilic catalysis in the serine protease mechanism

Specific and nonspecific thionester substrates for alpha-chymotrypsin and subtilisin Carlsberg have been synthesized and the kinetic parameters for their enzyme-catalyzed hydrolyses measured. Despite equal nonenzymic reactivities of ester-thionester pairs, each thionester is considerably less reactive toward enzymic hydrolysis, the difference being greatest for the specific substrates. The data support the operation of electrophilic catalysis by a hydrogen bond network at the carbonyl oxygen adjacent to the scissile bond of the substrate. The free energy of stabilization is 19 kJ mol-1 for a specific thionester substrate and will be higher for oxygen esters and amides. Chymotrypsin binds esters and thionesters about equally well, whereas subtilisin binds thionesters more tightly. This is consistent with continuous hydrogen bonding in the chymotrypsin mechanism and with a differential hydrogen bonding mechanism for subtilisin. A comparison of the relative rates of enzyme-catalyzed hydrolysis of ester and thionester substrates with their relative reactivities toward amines does not support an acyl histidine intermediate in the serine protease mechanism.     

Characterization of a zebrafish estrogen-sulfating cytosolic sulfotransferase: inhibitory effects and mechanism of action of phytoestrogens

Cytosolic sulfotransferases (STs) are generally thought to be involved in detoxification of xenobiotics, as well as homeostasis of endogenous compounds such as thyroid/steroid hormones and catecholamine hormones/neurotransmitters. We report here the identification and characterization of a zebrafish estrogen-sulfating cytosolic ST. The zebrafish ST was bacterially expressed, purified, and examined for enzymatic activities using a variety of endogenous compounds as substrates. Results showed that the enzyme displayed much higher activities toward two endogenous estrogens, estrone (E(1)) and 17beta-estradiol (E(2)), in comparison with thyroid hormones, 3,3',5-triiodothyronine (T(3)) and thyroxine (T(4)), dopamine, dihydroxyphenylalanine (Dopa), and dehydroepiandrosterone (DHEA). The kinetic parameters, K(m), and V(max), with estrogens and thyroid hormones as substrates were determined. The calculated V(max)/K(m) for E(1), E(2), T(3), and T(4) were, respectively, 31.6, 16.7, 1.5, and 0.8 nmol min(-1) mg(-1) microM(-1), indicating clearly the estrogens being preferred physiological substrates for the enzyme. The inhibitory effects of isoflavone phytoestrogens on the sulfation of E(2) by this zebrafish ST were examined. The IC(50) determined for quercetin, genistein, and daidzein were 0.7, 2.5, and 8 microM, respectively. Kinetic analyses revealed that the mechanism underlying the inhibition by these isoflavones to be of the competitive type.     

Resonance Raman as a direct probe for the catalytic mechanism of molybdenum oxotransferases

 Recent studies of human sulfite oxidase and Rhodobacter sphaeroides DMSO reductase have demonstrated the ability of resonance Raman to probe in detail the coordination environment of the Mo active sites in oxotransferases via Mo=O, Mo-S(dithiolene), Mo-S(Cys) or Mo-O(Ser), dithiolene chelate ring and bound substrate vibrations. Furthermore, the ability to monitor the catalytically exchangeable oxo group via isotopic labeling affords direct mechanistic information and structures for the catalytically competent Mo(IV) and Mo(VI) species. The results clearly demonstrate that sulfite oxidase cycles between cis–di-oxo-Mo(VI) and mono-oxo-Mo(IV) states during catalytic turnover, whereas DMSO reductase cycles between mono-oxo-Mo(VI) and des-oxo-Mo(IV) states. In the case of DMSO reductase, ¹⁸O-labeling experiments have provided the first direct evidence for an oxygen atom transfer mechanism involving an Mo=O species. Of particular importance is that the active-site structures and detailed mechanism of DMSO reductase in solution, as determined by resonance Raman spectroscopy, are quite different to those reported or deduced in the three X-ray crystallographic studies of DMSO reductases from Rhodobacter species. Received: 16 June 1997 / Accepted: 20 August 1997     

The mammalian cytosolic selenoenzyme thioredoxin reductase reduces ubiquinone. A novel mechanism for defense against oxidative stress

The selenoprotein thioredoxin reductase (TrxR1) is an essential antioxidant enzyme known to reduce many compounds in addition to thioredoxin, its principle protein substrate. Here we found that TrxR1 reduced ubiquinone-10 and thereby regenerated the antioxidant ubiquinol-10 (Q10), which is important for protection against lipid and protein peroxidation. The reduction was time- and dose-dependent, with an apparent K(m) of 22 microm and a maximal rate of about 12 nmol of reduced Q10 per milligram of TrxR1 per minute. TrxR1 reduced ubiquinone maximally at a physiological pH of 7.5 at similar rates using either NADPH or NADH as cofactors. The reduction of Q10 by mammalian TrxR1 was selenium dependent as revealed by comparison with Escherichia coli TrxR or selenium-deprived mutant and truncated mammalian TrxR forms. In addition, the rate of reduction of ubiquinone was significantly higher in homogenates from human embryo kidney 293 cells stably overexpressing thioredoxin reductase and was induced along with increasing cytosolic TrxR activity after the addition of selenite to the culture medium. These data demonstrate that the selenoenzyme thioredoxin reductase is an important selenium-dependent ubiquinone reductase and can explain how selenium and ubiquinone, by a combined action, may protect the cell from oxidative damage.