Kinetic studies on the broad-specificity <i>β</i>-<scp>d</scp>-glucosidase from pig kidney

Pócsi, István; Kiss, László

doi:10.1042/bj2560139

Cited by 25 publications

(7 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The generation of an sp 2 -hybridized C-1 carbon is expected to distort the ring into a half-chair conformation, with concordant distortions at the C-2 carbon and the ring oxygen. The C-6 carbon serves as an intramolecular "lever arm" through which the enzyme is able to force the pyranoside ring into a distorted half-chair conformation, as proposed previously (1,22,23).…”

Section: ␤-Glucosidase Mechanism and Transglycosylationmentioning

confidence: 85%

Catalytic Mechanism and Specificity for Hydrolysis and Transglycosylation Reactions of Cytosolic β-Glucosidase from Guinea Pig Liver

Hays¹,

VanderJagt²,

Bose³

et al. 1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

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...

show abstract

Section: ␤-Glucosidase Mechanism and Transglycosylationmentioning

confidence: 85%

Catalytic Mechanism and Specificity for Hydrolysis and Transglycosylation Reactions of Cytosolic β-Glucosidase from Guinea Pig Liver

Hays¹,

VanderJagt²,

Bose³

et al. 1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…This is in contrast to other b-glucosidases, which have wide substrate specificity, where glucose did not have an effect on substrate hydrolysis and the interaction with the hydrophobic aglycone was dominant, e.g. b-glucosidase from pig kidney (Po´csi & Kiss 1988), b-glucosidase from white clover (Po´csi et al 1989), b-glucosidase from cassava (Keresztessy et al 1994a). The strong inhibitory effect of glucono(1-5)lactone (K i ¼ 24 lM) proves that in the transition state the substrate is distorted into half chair conformation.…”

Section: Chemical Modification Of Carboxylate Groupsmentioning

confidence: 91%

Investigation of the active site of the extracellular β-D-glucosidase from Aspergillus carbonarius

Jäger

Kiss

2005

World J Microbiol Biotechnol

View full text Add to dashboard Cite

The catalytic amino acid residues of the extracellular b-D-glucosidase (b-D-glucoside glucohydrolase, EC 3.2.1.21) from Aspergillus carbonarius were investigated. The pH dependence curves gave apparent pK values of 2.8 and 5.93 for the free enzyme, and 2.24 and 6.14 for the enzyme-substrate complex using p-nitrophenyl-b-D-glucoside as substrate. Carbodiimide-and Woodward reagent K-mediated chemical modifications suggested that a carboxylate residue, located in the active centre, was fundamental in the catalysis. The pH dependence of inactivation revealed the involvement of a group with pK value of 4.61 in the modification reaction, proving that a carboxylate residue was modified. The A. carbonarius b-glucosidase was irreversibly inactivated by N-bromoacetyl-b-D-glucopyranosylamine. The active site specificity of the inactivation was proved by using the competitive inhibitor p-nitrophenyl-1-thio-b-D-glucopyranoside. pH Dependence studies of inactivation revealed that modification by N-bromoacetyl-b-D-glucopyranosylamine could be directed toward the carboxylate group acting as the catalytic nucleophile, as in the case of the carbodiimide and Woodward reagent K modifications.Abbreviations: pATP-Glc

show abstract

“…and various non-physiological aryl glycosides, including the pnitrophenyl and the 4-methylumbelliferyl derivatives of f6-Dglucose, /-D-galactose, a-L-arabinose, ,/-D-xylose, and fl-D-fucose [9,10]. Although the physiological function of the cytosolic broad-specificity fl-glucosidase has not been elucidated, the enzyme has been isolated from various sources [7,[11][12][13]] and its kinetic properties have been described extensively [14].…”

Section: Introductionmentioning

confidence: 99%

Human glucocerebrosidase catalyses transglucosylation between glucocerebroside and retinol

VanderJagt

Fry

Glew

1994

Biochemical Journal

View full text Add to dashboard Cite

The basal activity of human placental glucocerebrosidase is elevated 16-fold by n-pentanol when assayed using p-nitrophenyl beta-D-glucopyranoside (pNPGlc) as the beta-glucosidase substrate. This enhancement of activity is the result of the formation of a transglucosylation product, n-pentyl beta-D-glucoside, in rate-determining competition with the hydrolytic reaction. The transglucosylation product accounts for approximately 80% of the reaction product generated in the presence of n-pentanol (0.18 M) when either glucocerebroside or pNPGlc was used as the substrate. This stimulatory effect can be increased an additional 3-fold by the inclusion of phosphatidylserine (20 micrograms/ml) or sodium taurodeoxycholate (0.3%, w/v) in the incubation medium. In the presence of retinol, glucocerebrosidase also catalyses the synthesis of a novel lipid glucoside, retinyl glucoside, when either glucocerebroside or pNPGlc serves as the substrate. The reaction product was identified as retinyl beta-D-glucoside, based on its susceptibility to hydrolysis by almond beta-D-glucosidase and the subsequent release of equimolar amounts of retinol and glucose. The rate of retinyl-beta-glucoside formation is dependent on the concentration of retinol in the incubation medium, reaching saturation at approximately 0.3 mM retinol. Retinyl beta-D-glucoside is a substrate for two broad-specificity mammalian beta-glucosidases, namely the cytosolic and membrane-associated beta-glucosidases of guinea pig liver. However, retinyl beta-D-glucoside is not hydrolysed by placental glucocerebrosidase. These data indicate that the glucocerebrosidase-catalysed transfer of glucose from glucocerebroside to natural endogenous lipid alcohols, followed by the action of a broad-specificity beta-glucosidase on the transglucosylation product, could provide mammals with an alternative pathway for the breakdown of glucocerebroside to glucose and ceramide.

show abstract

Kinetic studies on the broad-specificity β-d-glucosidase from pig kidney

Cited by 25 publications

References 30 publications

Catalytic Mechanism and Specificity for Hydrolysis and Transglycosylation Reactions of Cytosolic β-Glucosidase from Guinea Pig Liver

Catalytic Mechanism and Specificity for Hydrolysis and Transglycosylation Reactions of Cytosolic β-Glucosidase from Guinea Pig Liver

Investigation of the active site of the extracellular β-D-glucosidase from Aspergillus carbonarius

Human glucocerebrosidase catalyses transglucosylation between glucocerebroside and retinol

Contact Info

Product

Resources

About