A range of 13 C-labeled carbohydrates containing C-O-C-C coupling pathways having different structures and dihedral angles has been prepared and used to identify structural factors affecting 3 J COCC , especially those across the O-glycosidic linkages of oligosaccharides. Model mono-and disaccharides were geometrically optimized using density functional methods, and scalar couplings involving carbon were calculated using a similar approach coupled with finite-field perturbation theory. Experimental and calculated 3 J COCC values were in close agreement, thus allowing use of the latter to better define the effect of carbohydrate structure on 3 J COCC magnitude. In addition to dihedral angle, the disposition of terminal electronegative substituents along the C-O-C-C coupling pathway significantly affects 3 J COCC values, and structural motifs have been identified where these effects may be encountered in oligosaccharides. A simple Karplus equation for trans-O-glycoside 3 J COCC values is proposed and has been applied in the reanalysis of trans-O-glycosidic couplings in 13 Clabeled methyl β-lactoside and sucrose. The behavior of trans-O-glycosidic 2 J COC and 3 J COCH values, which provide structural information complementary to that derived from 3 J COCC values, is also discussed.

No abstract

Bacterial isolates from the roots of wheat (rhizoplane) were more active in oxidizing glucose and alanine than cultures isolated from rhizosphere and non-rhizosphcre soils. In general, metabolic activity was greater with alanine than with glucose. Over one third of the cultures tested were capable of dissolving insoluble phosphorus in the form of CaHPO4 but the roots did not appear to exert a selective effect on these forms. However, the phosphate-solubilizing organisms from the rhizoplane were also the most active in oxidizing glucose and alanine. Those from the rhizosphere soil were intermediate in this respect. By far the majority of these phosphate-dissolving bacteria were in the nutritional group requiring unknown substances in yeast and soil extracts for optimal growth. It was suggested that although these bacteria were not preferentially stimulated in the root zone, their large numbers and their greater metabolic activity may contribute significantly to the phosphate economy of the plant.

No abstract

Cytosolic ␤-glucosidase (CBG) from mammalian liver is known for its broad substrate specificity and has been implicated in the transformation of xenobiotic glycosides. CBG also catalyzes a variety of transglycosylation reactions, which have been been shown with other glycosylhydrolases to function in synthetic and genetic regulatory pathways. We investigated the catalytic mechanism, substrate specificity, and transglycosylation acceptor specificity of guinea pig liver CBG by several methods. These studies indicate that CBG employs a two-step catalytic mechanism with the formation of a covalent enzyme-sugar intermediate and that CBG will transfer sugar residues to primary hydroxyls and equatorial but not axial C-4 hydroxyls of aldopyranosyl sugars. Kinetic studies revealed that correction for transglycosylation reactions is necessary to derive correct kinetic parameters for CBG. Further analyses revealed that for aldopyranosyl substrates, the activation energy barrier is affected most by the presence of a C-6 carbon and by the configuration of the C-2 hydroxyl, whereas the binding energy is affected modestly by the configuration and substituents at C-2, C-4, and C-5. These data indicate that the transglycosylation activity of CBG derives from the formation of a covalently linked enzymesugar intermediate and that the specificity of CBG for transglycosylation reactions is different from its specificity for hydrolysis reactions.A distinguishing feature of the cytosolic ␤-glucosidase (CBG) 1 of mammalian liver (EC 3.2.1.21) is its broad substrate specificity. The enzyme hydrolyzes ␤-D-galactopyranosides, ␤-D-fucopyranosides, ␤-D-xylopyranosides, and ␣-L-arabinopyranosides, in addition to ␤-D-glucopyranosides (1). The enzyme also catalyzes transglycosylation reactions in which a sugar residue is transferred from a substrate molecule to an acceptor to form a new glycoside (2). These properties are consistent with the fact that CBG is a configuration-retaining glycosidase (3). Collectively, these data suggest that the catalytic mechanism of CBG consists of a double-displacement reaction involving the formation of a stable enzyme-sugar intermediate, as originally proposed for configuration-retaining glycosidases by Koshland (4).Additional evidence of a two-step catalytic mechanism for CBG was derived from studies performed with the inhibitor, Br-conduritol-␤-epoxide, which is an irreversible, active sitedirected inactivator of the enzyme (5). However, studies with Escherichia coli ␤-galactosidase and human glucocerebrosidase have shown that the mechanism of inactivation by related conduritol epoxide compounds differs from the catalytic mechanisms of these two enzymes. For both glucocerebrosidase and ␤-galactosidase, the amino acid residue identified as the catalytic nucleophile by labeling with conduritol epoxides was later demonstrated to be incorrect by site-directed mutagenesis experiments (6, 7). The introduction of 2-deoxy-2-fluoro glycoside inhibitors by Withers and co-workers (8, 9) provided true mechanism-based i...