Broad-specificity Beta-glucosidase Research Articles

Cytosolic Pyridoxine-β-d-Glucoside Hydrolase from Porcine Jejunal Mucosa: PURIFICATION, PROPERTIES, AND COMPARISON WITH BROAD SPECIFICITY β-GLUCOSIDASE

During studies of the nutritional utilization of pyridoxine 5'-beta-D-glucoside, a major form of vitamin B6 in plants, we detected two cytosolic beta-glucosidases in jejunal mucosa. As expected, one was broad specificity beta-glucosidase that hydrolyzed aryl beta-D-glycosides but not pyridoxine beta-D-glucoside. We also found a previously unknown enzyme, designated pyridoxine-beta-D-glucoside hydrolase, that efficiently hydrolyzed pyridoxine beta-D-glucoside. These were separated and purified as follows: broad specificity beta-glucosidase 1460-fold and pyridoxine-beta-D-glucoside hydrolase 36,500-fold. Purified pyridoxine-beta-D-glucoside hydrolase did not hydrolyze any of the aryl glycosides tested but did hydrolyze cellobiose and lactose. Pyridoxine-beta-D-glucoside hydrolase exhibited a pH optimum of 5.5 and apparent molecular mass of 130 kDa by SDS-polyacrylamide gel electrophoresis and 160 kDa by nondenaturing gel filtration, in contrast to 60 kDa for native and denatured broad specificity beta-glucosidase. Glucono-delta-lactone was a strong inhibitor of both enzymes. Ionic and nonionic detergents were inhibitory for each enzyme. Conduritol B epoxide, a potent inhibitor of lysosomal acid beta-glucosidase, inhibited pyridoxine-beta-D-glucoside hydrolase but not broad specificity beta-glucosidase, but both were inhibited by the mechanism-based inhibitor 2-deoxy-2-fluoro-beta-D-glucosyl fluoride. Our findings indicate major differences between these two cytosolic beta-glucosidases. Studies addressing the role of vitamin B6 nutrition in regulating the activity and its consequences regarding pyridoxine glucoside bioavailability are in progress.

Human glucocerebrosidase catalyses transglucosylation between glucocerebroside and retinol

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.

Hydrolysis of Pyridoxine-5'- -d-Glucoside by a Broad-Specificity -Glucosidase from Mammalian Tissues

Research was conducted to evaluate the ability of a broad-specificity beta-glucosidase in mammalian tissues to catalyze the hydrolytic release of free pyridoxine from pyridoxine-5'-beta-D-glucoside, a naturally occurring form of vitamin B6 in plant-derived foods. Activity was detected in liver and intestinal mucosa using tritiated pyridoxine glucoside as a substrate. In the rat and guinea pig, enzyme activity was greater in intestine than in liver or kidney while even greater activity was detected in human intestinal tissue. Reaction rates were, however, low in all tissues. Hydrolysis of the synthetic substrate 4-methylumbelliferyl-beta-D-glucoside was also greatest in intestinal tissue. The characteristics of the enzymatic hydrolysis of pyridoxine glucoside to pyridoxine included: (i) most activity in the soluble tissue fraction, (ii) a pH optimum of approximately 6.0, and (iii) inhibition caused by the addition of sodium taurocholate. These characteristics are very similar to those of the broad-specificity beta-glucosidase in mammalian tissues with respect to the hydrolysis of a variety of naturally occurring and synthetic substrates. The apparent Km was greater than 2 mM for pyridoxine glucoside hydrolysis by intestinal preparations of each species, which is much greater than expected intestinal concentrations derived from dietary sources. In vivo studies have indicated that the intestine is involved in the metabolic utilization of dietary pyridoxine glucoside. The results observed here suggest that an alternate process, possibly involving intestinal microorganisms, may also be involved in the in vivo hydrolysis of pyridoxine glucoside.

Kinetic analysis of the interaction of alkyl glycosides with two human β-glucosidases

This paper addresses the similarities and differences in the topology of the catalytic centres of human liver cytosolic beta-glucosidase and placental lysosomal glucocerebrosidase, and utilizes well-documented reversible active-site-directed inhibitors. This comparative kinetic study was performed mainly to decipher the chemical and structural nature of the active site of the cytosolic beta-glucosidase, whose physiological function is unknown. Specifically, analysis of the effects of a family of alkyl beta-glucosides consistently displayed 100-250-fold lower inhibition constants with the cytosolic broad-specificity beta-glucosidase compared with the placental glucocerebrosidase; for example, with octyl beta-D-glucoside the Ki values were 10 microM and 1490 microM for the cytosolic and lysosomal beta-glucosidases respectively. Furthermore the higher affinity of the cytosolic beta-glucosidase than glucocerebrosidase for the amphipathic alkyl beta-D-glucosides was validated by the greater increase in the free energy of binding with increasing alkyl chain length [delta delta G0 (K,)/CH2: lysosomal enzyme, 2.01 kJ/mol (480 cal/mol); cytosolic enzyme, 3.05 kJ/mol (730 cal/mol)]. The implications of the presence of highly non-polar domains in the active site of the cytosolic beta-glucosidase are discussed with regard to its potential physiological substrates.

Hydrolysis of a naturally occurring beta-glucoside by a broad-specificity beta-glucosidase from liver.

We have isolated from guinea-pig liver a broad-specificity beta-glucosidase of unknown function that utilizes as its substrate non-physiological aryl glycosides (e.g. 4-methylumbelliferyl beta-D-glucopyranoside, p-nitrophenyl beta-D-glucopyranoside). The present paper documents that this enzyme can be inhibited by various naturally occurring glycosides, including L-picein, dhurrin and glucocheirolin. In addition, L-picein, which acts as a competitive inhibitor of the broad-specificity beta-glucosidase (Ki 0.65 mM), is also a substrate for this enzyme (Km 0.63 mM; Vmax. 277,000 units/mg). Heat-denaturation, kinetic competition studies, chromatographic properties and pH optima all argue strongly that the broad-specificity beta-glucosidase is responsible for the hydrolysis of both the non-physiological aryl glycosides and L-picein. This paper demonstrates that beta-glucosidase can catalyse the hydrolysis of a natural glycoside, and may provide a key to understanding the function of this enigmatic enzyme. A possible role in the metabolism of xenobiotic compounds is discussed.

Purification and characterization of a cytosolic broad specificity beta-glucosidase from human liver.

A cytoplasmic beta-glucosidase has been isolated and purified 9,000-fold to homogeneity from the liver of a case of type 1 Gaucher's disease to a specific activity of 400,000 nmol/h/mg of protein. Although markedly elevated above control levels in this case of adult Gaucher's disease, the activity of this cytosolic liver enzyme was found to be markedly deficient in two cases of neurologic Gaucher's disease. The purification scheme employs QAE-Sephadex, DE52 cellulose, CM-Sephadex, hydroxylapatite, and Cibacron blue-Sepharose chromatography, and preparative isoelectric focusing. The beta-glucosidase preparations isolated from the liver of the case of adult Gaucher's disease and control liver have similar physical properties. Both enzymes have a molecular weight of approximately 53,000, sw,20 of 4.3, pI of 4.5-4.6, a pH optimum between 5 and 6, and a high affinity for 4-methylumbelliferyl-beta-D-glucopyranoside (Km = 0.06-0.07 mM). The enzymes from both sources also have a broad specificity and will hydrolyze the 4-methylumbelliferyl derivatives of beta-D-galactose, beta-D-fucose, beta-D-xylose, and alpha-L-arabinose in addition to several aryl-galactosides and steroid-glucosides. The cytoplasmic beta-glucosidase will not hydrolyze glucocerebroside and shows no cross-reactivity with antibodies prepared against lysosomal glucocerebrosidase. Both cytoplasmic beta-glucosidase and glucocerebrosidase will hydrolyze 17 beta-estradiol-17'-beta-D-glucose, and the activity of both enzymes on this substrate is increased more than 15-fold in the presence of the Gaucher spleen heat-stable factor. The role of this cytoplasmic beta-glucosidase in the etiology of Gaucher's disease and its possible relationship to lysosomal glucocerebrosidase are discussed.