[Common properties and differences in the mechanism of action of glycoside hydrolases].
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Glycoside Hydrolases↗
Biomedical subjects
G Legler
Publications and source records attributed to G Legler.
Synthesis of 5-amino-5-deoxy-D-galactopyranose and 1,5-dideoxy-1,5-imino-D-galactitol, and their inhibition of alpha- and beta-D-galactosidases.
A 12-step route is presented starting from 1,2:5,6-di-O-isopropylidene-alpha-D-glucofuranose for the preparation of the title compounds and their L-altro analogues. Their synthesis is based on the reduction with Raney nickel of a protected 5-hydroxyimino derivative of L-arabino-hexofuranos-5-ulose, with the following improvements for the preparation of a D-galactofuranose derivative: oxidation at C-3 with pyridinium dichromate-acetic anhydride, stereospecific reduction of a 3-O-acetyl-hex-3-enofuranose intermediate to the D-gulo derivative, and inversion at C-3 of its 3-tosylate with tetrabutylammonium acetate in chlorobenzene. alpha-D-Galactosidase from coffee beans and from Escherichia coli and beta-D-galactosidase from E. coli and Aspergillus wentii were inhibited with Ki values that ranged from 0.0007 to 8.2 microM. Formation of the enzyme-inhibitor complexes with the D-galactose analogue was on the time-scale of minutes, whereas the D-galactitol analogue showed a slow approach to the inhibition only with alpha-D-galactosidase from coffee beans and beta-D-galactosidase from A. wentii. N-Alkylation of the D-galactitol analogue was detrimental to the inhibition except for beta-D-galactosidase from E. coli and beta-D-glucosidase from almonds, but, even with these enzymes, the observed affinity enhancements were 10(2) to 10(3)-times smaller than those of N-alkylated D-galactosylamine and D-glucosylamine.
Aspergillus↗
Active-site-directed inactivation of human liver alpha-L-fucosidase by conduritol C trans-epoxide.
Conduritol C trans-epoxide was found to inactivate human liver alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51), exhibiting an apparent dissociation constant of 43 mM. The cis-isomer of the inactivator had no apparent effect on the enzyme's activity. The pH profile for the inactivation yielded two apparent pK values of approx. 3.7 and 6.1 alpha-L-Fucose (a competitive inhibitor) was effective in protecting the enzyme from inactivation. These results are consistent with a requirement for two amino acid side chains at the active site involved in the reaction of the enzyme with conduritol C trans-epoxide.
Binding Sites↗
Human acid beta-glucosidase. Use of conduritol B epoxide derivatives to investigate the catalytically active normal and Gaucher disease enzymes.
Human acid beta-glucosidase (glucosylceramidase; EC 3.2.1.45) cleaves the glycosidic bonds of glucosyl ceramide and synthetic beta-glucosides. Conduritol B epoxide (CBE) and its brominated derivative are mechanism-based inhibitors which bind covalently to the catalytic site of acid beta-glucosidase. Procedures using brominetritiated CBE and monospecific anti-human placental acid beta-glucosidase IgG were developed to determine the molar concentrations of functional acid beta-glucosidase catalytic sites in pure placental enzyme preparations from normal sources; kcat values then were calculated from Vmax = [Et]kcat using glucosyl ceramide substrates with dodecanoyl (2135 +/- 45 min-1) and hexanoyl (3200 +/- 410 min-1) fatty acid acyl chains and 4-alkyl-umbelliferyl beta-glucoside substrates with methyl (2235 +/- 197 min-1), heptyl (1972 +/- 152 min-1), nonyl (2220 +/- 247 min-1), and undecyl (773 +/- 44 min-1) alkyl chains. The respective kcat values for acid beta-glucosidase in a crude normal splenic preparation were about 60% of these values. In comparison, the kcat values of the mutant splenic acid beta-glucosidase from two Type 1 Ashkenazi Jewish Gaucher disease (AJGD) patients were about 1.5-3-fold decreased and had Km values for each substrate which were similar to those for the normal acid beta-glucosidase. The interaction of the normal and Type 1 AJGD enzymes with CBE in a 1:1 stoichiometry conformed to a model with reversible EI complexes formed prior to covalent inactivation. With CBE, the equal kmax values (maximal rate of inactivation) for the normal (0.051 +/- 0.009 min-1) and Type 1 AJGD (0.058 +/- 0.016 min-1) enzymes were consistent with the minor differences in kcat. In contrast, the Ki value (dissociation constant) (839 +/- 64 microM) for the Type 1 AJGD enzymes was about 5 times the normal Ki value (166 +/- 57 microM). These results indicated that the catalytically active Type 1 AJGD acid beta-glucosidase had nearly normal hydrolytic capacity and suggested an amino acid substitution in or near the acid beta-glucosidase active site leading to an in vivo instability of the mutant enzymatic activity.
Gaucher Disease↗
Purification and characterization of a neutral processing mannosidase from calf liver acting on (Man)9(GlcNAc)2 oligosaccharides.
A processing mannosidase acting on (Man)9(GlcNAc)2 oligosaccharides, Man9 mannosidase, has been purified 2190-fold from calf liver crude microsomes by a four-step procedure involving (a) differential salt/detergent extraction, (b) affinity chromatography on AH-Sepharose 4B with N-5-carboxypentyl-1-deoxymannojirimycin as ligand, (c) ConA-Sepharose and (d) DEAE-Sephacel chromatography. (Man)9 mannosidase has a subunit molecular mass of 56 kDa and does not bind to ConA-Sepharose, indicating the absence of high-mannose oligosaccharides. The enzyme has a pH optimum close to pH 6.0 and requires divalent cations for activity, Ca2+ being most effective. It is inhibited by 1-deoxymannojirimycin (dMM), N-methyl-dMM and N-5-carboxypentyl-dMM with Ki = 7 microM, 75 microM, and 140 microM, respectively. Man9 mannosidase cleaves three of the four alpha 1,2-linked mannose residues from the (Man)9(GlcNAc)2 oligosaccharide, does not hydrolyse the remaining (Man)6(GlcNAc)2 structure and is not active against aryl alpha-mannosides. This pronounced substrate specificity points to the participation of Man9 mannosidase in the N-linked processing pathway and, in addition, clearly distinguishes this enzyme from the mannosidases reported previously. As Man9 mannosidase appears to act in the processing sequence immediately after the three glucose residues have been removed from the (Glc)3(Man)9(GlcNAc)2 intermediate, we assume that the enzyme is located in the endoplasmic reticulum.
1-Deoxynojirimycin↗
Characterization of calf liver glucosidase I and its inhibition by basic sugar analogs.
Glucosidase I, the enzyme catalyzing the first step of N-linked oligosaccharide processing, has been purified from calf liver crude membranes [H. Hettkamp, G. Legler, and E. Bause, (1984) Eur. J. Biochem. 142, 85-90]. Binding experiments with concanavalin A-Sepharose suggest that glucosidase I is a glycoprotein with high-mannose carbohydrate chain(s). The enzyme has a subunit molecular mass of approximately 83 kDa and specifically hydrolyzes the terminal alpha-1,2-linked glucose residue from the natural Glc3-Man9-GlcNAc2 oligosaccharide. Studies with a variety of substrates modified in the aglycon moiety suggest that the Glc2 branch rather than the more distant domains of the substrate molecule are important for binding and hydrolysis. Glucosidase I does not require metal ions for activity and is strongly inhibited by 1-deoxynojirimycin (dNM) and its N-alkyl derivatives. Ki values range from 0.07 microM for N-methyl-dNM to 1.0 microM for dNM, measured at the pH-optimum of enzyme activity. The pH dependence of inhibition indicates that the cationic form of the inhibitors is the active species. Comparison of the Ki for N-decanoyl-dNM (approximately 70 microM) with that of N-decyl-dNM (approximately 0.4 microM) suggests that electrostatic interactions at the catalytic site of the enzyme are important for inhibitor binding. 1-Deoxymannojirimycin, previously assumed to be a specific mannosidase inhibitor, as well as its N-methyl and N-5-carboxypentyl derivatives, inhibit glucosidase I with Ki values around 190, 17, and 100 microM, respectively. This apparent lack of specificity shows that in vivo experiments on N-glycoprotein processing as well as the interpretation of results with these mannosidase inhibitors may give misleading results when these compounds are used in the millimolar range.
1-Deoxynojirimycin↗
Human acid beta-glucosidase: isolation and amino acid sequence of a peptide containing the catalytic site.
Human acid beta-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) cleaves the glucosidic bonds of glucosylceramide and synthetic beta-glucosides. The deficient activity of this hydrolase is the enzymatic defect in the subtypes and variants of Gaucher disease, the most prevalent lysosomal storage disease. To isolate and characterize the catalytic site of the normal enzyme, brominated 3H-labeled conduritol B epoxide (3H-Br-CBE), which inhibits the enzyme by binding covalently to this site, was used as an affinity label. Under optimal conditions 1 mol of 3H-Br-CBE bound to 1 mol of pure enzyme protein, indicating the presence of a single catalytic site per enzyme subunit. After V8 protease digestion of the 3H-Br-CBE-labeled homogeneous enzyme, three radiolabeled peptides, designated peptide A, B, or C, were resolved by reverse-phase HPLC. The partial amino acid sequence (37 residues) of peptide A (Mr, 5000) was determined. The sequence of this peptide, which contained the catalytic site, had exact homology to the sequence near the carboxyl terminus of the protein, as predicted from the nucleotide sequence of the full-length cDNA encoding acid beta-glucosidase.
Amino Acid Sequence↗
Human acid beta-glucosidase: affinity purification of the normal placental and Gaucher disease splenic enzymes on N-alkyl-deoxynojirimycin-sepharose.
Two sepharose-bound 1-deoxynojirimycin N-alkyl derivatives, N-(9-carboxynonyl)- and N-(11-carboxyundecyl)-deoxynojirimycin, were used for the affinity purification of acid beta-glucosidase (beta-Glc) from normal and type-1 Ashkenazi Jewish Gaucher disease (AJGD) sources. The capacities of these nondegradable inhibitor supports were 0.5 and 0.75 mg of normal beta-Glc/ml of settled gel, respectively. The purified normal enzyme (14-18% yield) had a specific activity of 1.6 X 10(6) nmol/h/mg protein and was homogeneous as evidenced by a single protein species of Mr = 67,000 on sodium dodecylsulfate-polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography (HPLC). Microsequencing demonstrated a single N terminus, and the sequence of the first 22 N-terminal amino acids was colinear with that predicted from the beta-Glc cDNA. Amino acid composition analyses of beta-Glc revealed a high content (35%) of hydrophobic amino acids. The N-decyl-deoxynojirimycin support facilitated the purification of the residual enzyme from type-1 AJGD spleen to about 7,500-fold in four steps with a yield of about 11%. These new affinity supports provided improved stability, capacity and/or specificity compared to other affinity or HPLC methods for purifying this lysosomal glycosidase.
1-Deoxynojirimycin↗
On the chemical basis of the Lowry protein determination.
The copper-catalyzed oxidation of peptides and proteins by phosphomolybdic/phosphotungstic acid (Folin phenol reagent) was studied with respect to redox stoichiometry of color formation and nature of the oxidation products. From peptides without reducing side chains two reducing equivalents were transferred under ideal conditions to Mo6+/W6+ for each unit of tetradentate copper complex with concomitant formation of an imino peptide. Tyrosine and tryptophan side chains contributed four additional reducing equivalents. Oxidation of proline-containing peptides was greatly impaired as judged from color formation due to the interference of the imino acid with complex formation. Reaction of the oxidized peptides with 2,4-dinitrophenyl (DNP)-hydrazine gave a peptide amine and the DNP-hydrazone of a 2-oxoacyl peptide. The oxidation products from tetraalanine were identified as dialanine amide and pyruvoylalanine DNP-hydrazone. From the time course of the development of the blue color on reduction of Folin reagent with tetraalanine it was inferred that the reaction consisted of an initial (less than 5 s) oxidation to a Cu3+ peptide complex followed by slow changes in absorbance, especially above 0.2 mM. Due to these complications the two-electron stoichiometry has to be considered only as a limiting case for peptide concentrations below 0.02 mM.
Chemical Phenomena↗
The mannosidase inhibitors 1-deoxymannojirimycin and swainsonine have no effect on the biosynthesis and infectivity of Rous sarcoma virus.
The effects of inhibitors, which interfere with oligosaccharide trimming by blocking mannosidases, on the processing and export of the viral glycoproteins of Rous sarcoma virus (RSV), have been studied. 1-Deoxymannojirimycin (DIM) prevents removal of mannose residues from the Man9 (GlcNAc)2 oligosaccharide whereas swainsonine (SW) blocks at a later stage resulting in the formation of so-called hybrid oligosaccharides. Under a regime of these inhibitors, proteolytic cleavage of the viral glycoprotein precursor can still occur to yield aberrant glycoprotein products, gp75DIM/gp30DIM and gp80SW/gp30SW. Virus particles carrying these aberrant viral glycoproteins are released from inhibitor-treated cultures in normal amounts and these virions are fully infectious. Thus blocking oligosaccharide trimming at the stages described here or, using different inhibitors, at different stages as described previously (J. V. Bosch and R. T. Schwarz, Virology 132, 95-109 (1984)), does not have any influence on the infectivity of Rous sarcoma virus.
1-Deoxynojirimycin↗
Goat liver beta-mannosidases: molecular properties, inhibition and inactivation of the lysosomal and nonlysosomal forms.
The two caprine hepatic beta-mannosidases have been partially purified and their properties have been compared. The lysosomal beta-mannosidase A had an apparent molecular weight of 127,000 +/- 10,000 and an isoelectric point of pH 6-7. Its activity was unaffected by incubation with Triton X-100 (0.1%) and cysteine (20 mM) and it hydrolyzed the presumed natural substrates, Man(beta 1-4)GlcNAc and Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc. The nonlysosomal beta-mannosidase B had an apparent molecular weight of 43,000 +/- 2,000 and an isoelectric point of pH 5.5. beta-Mannosidase B was activated by Triton X-100 (0.1%) and was inhibited by cysteine (20 mM). Hydrolysis of Man(beta 1-4)GlcNAc, but not of Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc, followed incubation with beta-mannosidase B. 1,5-Dideoxy-1,5-imino-D-mannitol did not inhibit the A enzyme and only feebly (Ki = 0.3 mM) inhibited the B enzyme; beta-D-mannopyranosylmethyl p-nitrophenyl triazene did not inactivate either enzyme but 1,2-anhydro-1,2,3,5,6/4-cyclohexane hexol inactivated the B enzyme only. The radical mechanistic differences between the two enzymes argue against their having the same genetic origin.
Animals↗
Cell type dependent inhibition of transport of cathepsin D in HepG2 cells and fibroblasts exposed to deoxy-manno-nojirimycin and deoxynojirimycin.
The synthesis, transport and processing of lysosomal enzymes was examined in human hepatoma HepG2 cells and in human fibroblasts exposed to the Golgi alpha-mannosidase I inhibitor 1-deoxy-manno-nojirimycin. In HepG2 cells cathepsin D, beta-hexosaminidase and arylsulfatase B synthesized in the presence of 5 mM 1-deoxy-manno-nojirimycin contained exclusively endo-beta-N-acetylglucosaminidase H-cleavable oligosaccharides, indicating that alpha-mannosidase I had been inhibited efficiently. The proteolytic processing of intracellularly retained cathepsin D was retarded and the fraction of secreted cathepsin D was increased two-fold. In fibroblasts neither segregation nor maturation of cathepsin D were affected by 1-deoxy-manno-nojirimycin in spite of the inhibition of oligosaccharide processing. In the presence of the glucosidase I inhibitor 1-deoxynojirimycin, the precursor of cathepsin D (larger by about 1 kDa than the secreted form) accumulated transiently in light membranes in HepG2 cells. Release from the site of accumulation was accompanied by a decrease in size by about 1 kDa. This change was attributed to the removal of glucose residues. In fibroblasts the transient accumulation of larger precursors in the presence of 1-deoxynojirimycin was more pronounced than in HepG2 cells. The differential effects of alpha-mannosidase I and glucosidase I inhibitors on the transport of cathepsin D in HepG2 cells and fibroblasts may indicate that different intermediates in the biosynthetic pathway of asparagine-linked oligosaccharides participate in the transport of lysosomal enzymes in the two cell types.
1-Deoxynojirimycin↗
Glucosylceramidase from calf spleen. Characterization of its active site with 4-n-alkylumbelliferyl beta-glucosides and N-alkyl derivatives of 1-deoxynojirimycin.
The beta-glucosides of 4-heptyl-, -nonyl-, and -undecylumbelliferone were synthesized and their substrate properties studied with calf spleen glucosylceramidase. Self-association of the free long chain alkylumbelliferones in aqueous buffer was inferred from their low fluorescence in the absence and strongly enhanced fluorescence in the presence of detergents. Association of the higher alkylumbelliferyl glucosides with detergent micelles was indicated by the influence of detergent on solubility and on enzyme activity which differed markedly between the methyl and the higher alkyl substrates. Compared to 4-methylumbelliferyl beta-glucoside their Km was 14 to 23 times smaller and Vmax/Km 20 to 30 times larger with no significant difference between the nonyl and undecyl derivatives. The enzyme was inhibited by 1-deoxynojirimycin (1,5-dideoxy-1,5-imino-D-glucitol, dNM) and a series of its N-alkyl derivatives with Ki-values that ranged from 390 microM for the parent compound to 330 microM for the butyl derivative and 0.08 microM for the tetradecyl derivative. The biphasic linear plot of - RT X 1n [Ki/Ki (dNM)] vs. chain length is interpreted in terms of an aglycon binding site that has an extended hydrophobic region starting at about 5 carbon atoms from the catalytic site. dNM inhibited greater than or equal to 10(3) times better than D-glucose, and N-decanoyl-dNM was a very weak inhibitor compared to N-decyl-dNM. It is concluded that the formation of an ion pair consisting of the protonated dNM derivative and an essential carboxylate at the catalytic site makes a large contribution to the binding energy. Strong shielding of this site from the aqueous environment is indicated by identical effects of ionic strength on Km and Ki.
1-Deoxynojirimycin↗
Influence of asparagine-linked oligosaccharides on tumor cell recognition in the mixed lymphocyte reaction.
The ability of an Ia+ B cell lymphoma, AKTB-1b, to stimulate thymocytes in the allogeneic mixed lymphocyte reaction is dependent on its prior treatment with either swainsonine or deoxynojirimycin, two inhibitors of the processing of asparagine-linked oligosaccharides. In the absence of drug treatment, the tumor cells fail to stimulate thymocytes, whereas pretreatment of the tumor cells with either drug results in a five- to 10-fold increase in their ability to induce thymocyte proliferation. Drug-treated AKTB-1b stimulates thymocytes at levels comparable to those obtained with allogeneic splenocytes. In contrast, the untreated lymphoma does stimulate splenic lymphocytes, and pretreatment with either inhibitor only marginally increases the response. Genetic studies demonstrate that the thymocyte response is still H-2 locus restricted and can be blocked by monoclonal antibodies against two tumor cell major histocompatibility antigens, H-2K and I-A. Drug treatment does not change cell surface I-A expression, and H-2K levels are apparently decreased one-third by deoxynojirimycin but are not affected by swainsonine. To verify that the drug protocol used was capable of altering the glycoconjugates of membrane-associated proteins, the endo-beta-N-acetylglucosaminidase H (endo H)-sensitivity of immunopurified H-2K and I-A was analyzed by SDS-PAGE. These studies demonstrated that swainsonine treatment does result in cell surface expression of glycoconjugates with altered oligosaccharide moieties. Likewise, deoxynojirimycin treatment results in the cell surface expression of an I-A alpha polypeptide with altered oligosaccharide chains while only marginally affecting H-2K and not affecting the I-A beta chain. An intracellular form of the I-A beta chain sensitive to endo H digestion in the presence of deoxynojirimycin is not detectable at the cell surface. Neuraminidase-digested AKTB-1b are also capable of stimulating allogeneic thymocytes. These studies demonstrate that changes in the glycosylation state of the tumor cell can markedly influence its recognition by allogeneic lymphocytes, and further, that different T cell populations differ in their response to such changes.
1-Deoxynojirimycin↗
Gaucher disease types 1, 2, and 3: differential mutations of the acid beta-glucosidase active site identified with conduritol B epoxide derivatives and sphingosine.
To elucidate the genetic heterogeneity in Gaucher disease, the residual beta-glucosidase in cultured fibroblasts from affected patients with each of the major phenotypes was investigated in vitro and/or in viable cells by inhibitor studies using the covalent catalytic site inhibitors, conduritol B epoxide or its bromo derivative, and the reversible cationic inhibitor, sphingosine. These studies delineated three distinct groups (designated A, B, and C) of residual activities with characteristic responses to these inhibitors. Group A residual enzymes had normal I50 values (i.e., the concentration of inhibitor that results in 50% inhibition) for the inhibitors and normal or nearly normal t1/2 values for conduritol B epoxide. All neuronopathic (types 2 and 3) and most non-Jewish nonneuronopathic (type 1) patients had group A residual activities and, thus, could not be distinguished by these inhibitor studies. Group B residual enzymes had about four- to fivefold increased I50 values for the inhibitors and similarly increased t1/2 values for conduritol B epoxide. All Ashkenazi Jewish type 1 and only two non-Jewish type 1 patients had group B residual activities. The differences in I50 values between groups A and B also were confirmed by determining the uninhibited enzyme activity after culturing the cells in the presence of bromo-conduritol B epoxide. Group C residual activity had intermediate I50 values for the inhibitors and represented a single Afrikaner type 1 patient: this patient was a genetic compound for the group A (type 2) and group B (type 1) mutations. These inhibition studies indicated that: Gaucher disease type 1 is biochemically heterogeneous, neuronopathic and non-Jewish nonneuronopathic phenotypes cannot be reliably distinguished by these inhibitor studies, and the Ashkenazi Jewish form of Gaucher disease type 1 results from a unique mutation in a specific active site domain of acid beta-glucosidase that leads to a defective enzyme with a decreased Vmax.
Adolescent↗
Purification by affinity chromatography of glucosidase I, an endoplasmic reticulum hydrolase involved in the processing of asparagine-linked oligosaccharides.
Trimming glucosidase I and II have been solubilized from crude calf liver microsomes and partially enriched by a fractionated extraction procedure applying different concentrations of nonionic detergent and salt. The pH optimum of both enzymes was found to be close to 6.2, which discriminates them from hydrolases of lysosomal origin acting on p-nitrophenyl glycosides with the highest rate at more acidic pH. Glucosidase I and II and the nonspecific alpha-glucosidase(s) were inhibited by 1-deoxynojirimycin with median inhibitory concentration of 3 microM, 20 microM, 12 microM, respectively. Discrimination between these enzymes was strongly enhanced by N-alkylation of 1-deoxynojirimycin and formed the basis for the design of the affinity ligand. Glucosidase I has been purified to homogeneity by affinity chromatography on AH-Sepharose 4B with N-carboxypentyl-1-deoxynojirimycin as ligand. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed a subunit molecular mass of about 85 kDa. The molecular mass of the native enzyme, determined by gel chromatography, was approximately equal to 320-350 kDa, pointing to the association of subunits to a tetramer. Glucosidase I is rather stable when stored at 4 degrees C in the presence of detergent (t 1/2 approximately equal to 20 days) and showed high specificity for the hydrolysis of the terminal (alpha 1,2)-linked glucose residue in the natural substrate Glc3-Man9-(GlcNAc)2.
1-Deoxynojirimycin↗
Synthesis of 5-amino-5-deoxy-D-mannopyranose and 1,5-dideoxy-1,5-imino-D-mannitol, and inhibition of alpha- and beta-D-mannosidases.
The title compounds and the corresponding L-gulo derivatives were synthesised in 6 steps from benzyl 2,3:5,6-di-O-isopropylidene-alpha-D-mannofuranoside. The Ki values, determined from inhibition studies with alpha-D-mannosidases from jack beans, almonds, and calf liver, and beta-D-mannosidase from Aspergillus wentii, ranged from 70 to 400 microM for the mannitol derivative and from 1.2 to 20 microM for 5-amino-5-deoxy-D-mannopyranose, i.e., inhibition is 10(2)-10(4)-fold stronger than with D-mannose. Marked enhancement of inhibition with increasing pH is ascribed to the ionisation of a carboxyl group at the active site, forming an ion pair with the protonated inhibitor. The inhibition equilibrium between the jack-bean enzyme and the mannose derivative was approached slowly with kapp 2.0 X 10(5) M-1 X min-1. The mannose-derived inhibitor was also inhibitory against beta-D-glucosidases from almonds and Asp. wentii, with Ki values only 20-150-times larger than those for the inhibition of these enzymes by 5-amino-5-deoxy-D-glucopyranose. This moderate discrimination in binding of D-gluco and D-manno derivatives is in marked contrast to the high specificity shown by the glucosidase in catalysing the hydrolysis of mannosidases. A similar low specificity with respect to binding, combined with highly specific catalysis, was also seen with the mannosidases acting on inhibitors and substrates with the D-gluco configuration.
1-Deoxynojirimycin↗
Identification of an essential carboxylate group at the active site of lacZ beta-galactosidase from Escherichia coli.
[3H] Conduritol C cis-epoxide (1,2-anhydro-epi-inositol, I) was synthesized as an active-site-directed inhibitor for lacZ beta-galactosidase from Escherichia coli. A considerable kinetic isotope effect was noted in the reduction by [3H]NaBH4 of the p-benzoquinone-derived precursor for I. Complete loss of beta-galactosidase activity occurred on incorporation of 4 mol I/mol beta-galactosidase tetramer. The inhibitor was very labile in the denatured enzyme at pH greater than 8, implying the formation of an ester bond between I and a carboxylate at the active site. The radioactive material released from the labeled enzyme was identified as allo-inositol. The stereochemistry of the expoxide reaction (trans-diaxial ring opening) is thus the same as for beta-glucosidases with the corresponding epoxides. The binding site for I was identified as Glu-461 by the isolation and partial sequence analysis of a radioactive octapeptide from the cyanogen bromide and pepsin fragments of the labeled enzyme. A failure to determine the N-terminal amino acid of the labeled peptide is ascribed to the great reactivity of the esterified gamma-carboxyl group of its N-terminal Glu-461 which causes rapid cyclisation of this residue to pyroglutamate, even under weakly basic conditions. The participation of the carboxylate of Glu-461 in catalysis is discussed.
Binding Sites↗