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.     

Normalization of liver glucosylceramide levels in the "Gaucher" mouse by phosphatidylserine injection

A model of the human genetic disorder, Gaucher disease, can be rapidly generated in mice by the injection of emulsified glucosylceramide and an inhibitor of the lipid's hydrolase, conduritol B epoxide. The liver grows rapidly as it absorbs the load of lipid but the effect disappears as new glucosidase is formed and the load is hydrolyzed. This normalization process is accelerated by treatment with phosphatidylserine, which is a known stimulator of the enzyme. It is possible that injecting the phospholipid into Gaucher patients would have a therapeutic effect since it might help them utilize their residual glucosidase to destroy stored glycolipid.     

Use of activators and inhibitors to define the properties of the active site of normal and Gaucher disease lysosomal beta-glucosidase

Lysosomal beta-glucosidase ('glucocerebrosidase') in peripheral blood lymphocyte and spleen extracts from normal individuals and Ashkenazi-Jewish Gaucher disease type-1 patients were investigated using several modifiers of glucosyl ceramide hydrolysis. The negatively charged lipids, phosphatidylserine and taurocholate, had differential effects on the hydrolytic rates of the normal and Gaucher disease enzymes from either source. With the normal enzyme, either negatively charged lipid (up to 1 mmol/l) increased the reaction rates, while decreasing hydrolytic rates were obtained at greater concentrations. In comparison, the peak activities of the Gaucher enzymes were observed at about 2-3 mmol/l or 5-8 mmol/l of phosphatidylserine or taurocholate, respectively. These negatively charged lipids altered only the velocity of the reactions; the apparent Km values were not affected. Taurocholate or phosphatidylserine also facilitated the interaction of the normal enzyme with conduritol B epoxide, a covalent inhibitor of the catalytic site. Compared to the normal enzyme, the Ashkenazi-Jewish Gaucher type-1 enzyme required about 5-fold greater concentrations of conduritol B epoxide for 50% inhibition. Neutral or cationic acyl-beta-glucosides were found to be competitive or noncompetitive inhibitors of the enzymes, respectively. Alkyl beta-glucosides were competitive (or linear-mixed type) inhibitors of the normal splenic or lymphocyte enzyme with competitive inhibition constants (Ki) inversely related to the chain length. With octyl and dodecyl beta-glucoside nearly normal competitive Ki values were obtained with the splenic enzymes from Gaucher patients. These Ki values were not influenced by increasing phosphatidylserine or taurocholate concentrations. In contrast, the cationic lipids, sphingosyl-1-O-beta-D-glucoside (glucosyl sphingosine) and its N-hexyl derivative, were noncompetitive inhibitors whose apparent Ki values for the normal enzyme were 30 and 0.25 mumol/l, respectively. The Ki values for these sphingosyl glucosides were about increased 5 times for the Gaucher type-1 enzymes from Ashkenazi-Jewish Gaucher disease type-1 patients. The Ki values of glucosyl sphingosine for the normal or mutant enzymes were directly related to increasing concentrations of phosphatidylserine or taurocholate. This latter site appears to be specifically altered by a mutation in the structural gene for lysosomal beta-glucosidase in the Ashkenazi-Jewish form of type-1 Gaucher disease.     

Glucosylceramide modulates membrane traffic along the endocytic pathway

Glycosphingolipids are endocytosed and targeted to the Golgi apparatus, but are mistargeted to lysosomes in numerous sphingolipidoses. Substrate reduction therapy utilizes imino sugars to inhibit glucosylceramide synthase and potentially abrogate the effects of storage. Gaucher disease is a hereditary deficiency in glucocerebrosidase leading to glucosylceramide accumulation; however, Gaucher fibroblasts exhibited normal Golgi transport of lactosylceramide. To better understand the effects of glycosphingolipid accumulation on intracellular trafficking and the use of imino sugar inhibitors, we studied sphingolipid endocytosis in fibroblast and macrophage models for Gaucher disease. Treatment of fibroblasts or RAW macrophages with conduritol B epoxide, an inhibitor of lysosomal glucocerebrosidase, resulted in a change in the endocytic targeting of lactosylceramide from the Golgi to the lysosomes. Co-treatment of macrophages with conduritol B-epoxide and 12-25 microM N-butyldeoxygalactonojirimycin, an inhibitor of glycosphingolipid biosynthesis, prevented the mistargeting of lactosylceramide to the lysosomes and restored trafficking to the Golgi. Surprisingly, higher doses (>25 microM) of NB-DGJ induced targeting of lactosylceramide to the lysosomes, even in the absence of conduritol B-epoxide. These data demonstrate that both increases and decreases in glucosylceramide levels can dramatically alter the endocytic targeting of lactosylceramide and suggest a role for glucosylceramide in regulation of membrane transport.     

Gaucher disease: genetic heterogeneity within and among the subtypes detected by immunoblotting.

The genetic heterogeneity of Gaucher disease subtypes and variants was investigated by immunoblotting of fibroblast extracts. For these studies polyclonal and monoclonal antibodies were raised to acid beta-glucosidase preparations containing a single N-terminal amino acid sequence that was colinear with that encoded by the beta-Glc cDNAs. Three forms (Mr approximately equal to 67,000, 64,000-61,000, and 58,000) of cross-reacting immunologic material (CRIM) were observed in control individuals. Decreased amounts of the same CRIM forms were detected in most type 1 Gaucher disease patients, but single CRIM forms of variable molecular weight were observed in several non-Jewish type 1 variants. One or two CRIM forms of variable molecular weight were found in neuronopathic (type 2 and type 3) patients. The amount of CRIM was severely decreased in the majority of the type 2 and type 3 patients; one American black type 2 patient was CRIM negative. With this one exception, one CRIM form was detected in the cell-free culture media from all normal or Gaucher disease fibroblasts that had an Mr approximately 2,000 greater than the highest respective intracellular molecular-weight form. All intra- or extracellular CRIM forms were reduced to a single form after deglycosylation with N-Glycanase. In addition, the radioactivity from [3H]Br-conduritol B epoxide, a specific covalent inhibitor of beta-Glc, localized to the CRIM forms of beta-Glc on immunoblots. These results indicate that all subtypes and variants of Gaucher disease result from mutations that alter the stability and/or processing of beta-Glc. Furthermore, the heterogeneity of the CRIM patterns within and among the variants of Gaucher disease cause the diagnostic usefulness of immunoblotting to be restricted to those families in which the phenotype has been well established.     

An in situ study of beta-glucosidase activity in normal and gaucher fibroblasts with fluorogenic probes

Beta-glucosidase activity was evaluated in situ by means of fluorogenic probes in normal human fibroblasts and fibroblasts from homozygous carriers of the Gaucher trait. Probe internalization, targeting to lysosomes and post-cleavage probe retention were the primary concerns. Internalization and targeting were attempted by in situ photosensitized labilization of lysosomal membranes, lysosomotropic detergents and the use of low density lipid (LDL) or the receptor ligand apolipoprotein E (ApoE). Post-cleavage increase of fluorescence with fluoresceinyl (bis) betaglucopyranoside was appreciably above the rather large pre-cleavage emission. In cells incubated overnight with nonylumbelliferylbetaglucoside (UG9) in the presence of bovine serum albumin and in the absence of ApoE, the probe was dealt with as a cytotoxic agent, accumulating in a paranuclear cap, most likely comprising elements of the endoplasmic reticulum (ER) and Golgi apparatus. Targeting of UG9 to lysosomes occurred within 1 to 3 h of preincubation in the presence of ApoE. There was some evidence of specificity, as Gaucher fibroblasts exhibited weaker cleavage of UG9 (by 50 per cent or more) compared to normal fibroblasts, but in the Gaucher cells there was some residual beta-glucosidase activity. Cleavage of UG9 was nearly totally suppressed in Gaucher cells treated with the beta-glucosidase inhibitor, conduritol B epoxide, for 24 h to 7 days. Suppression in the control fibroblasts was evident but to a lesser degree. The in situ method of fluorogenic assay established for beta-glucosidase deficiency, is in principle applicable to enzyme deficiencies in other lysosomal storage diseases, or to evaluate enhanced enzyme activity following gene therapy.     

Properties of Neurons Derived from Induced Pluripotent Stem Cells of Gaucher Disease Type 2 Patient Fibroblasts: Potential Role in Neuropathology

Gaucher disease (GD) is caused by insufficient activity of acid β-glucosidase (GCase) resulting from mutations in GBA1. To understand the pathogenesis of the neuronopathic GD, induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from three GD type 2 (GD2) and 2 unaffected (normal and GD carrier) individuals. The iPSCs were converted to neural precursor cells (NPCs) which were further differentiated into neurons. Parental GD2 fibroblasts as well as iPSCs, NPCs, and neurons had similar degrees of GCase deficiency. Lipid analyses showed increases of glucosylsphingosine and glucosylceramide in the GD2 cells. In addition, GD2 neurons showed increased α-synuclein protein compared to control neurons. Whole cell patch-clamping of the GD2 and control iPSCs-derived neurons demonstrated excitation characteristics of neurons, but intriguingly, those from GD2 exhibited consistently less negative resting membrane potentials with various degree of reduction in action potential amplitudes, sodium and potassium currents. Culture of control neurons in the presence of the GCase inhibitor (conduritol B epoxide) recapitulated these findings, providing a functional link between decreased GCase activity in GD and abnormal neuronal electrophysiological properties. To our knowledge, this study is first to report abnormal electrophysiological properties in GD2 iPSC-derived neurons that may underlie the neuropathic phenotype in Gaucher disease.     

Enzymic effects of beta-glucosidase destruction in mice. Changes in glucuronidase levels.

1. The injection into mice of a single dose of conduritol B epoxide, a covalent inhibitor of glucosidases, quickly produced changes in tissue levels of beta-D-glucuronidase (EC 3.2.1.31). The specific activity of the enzyme decreased in liver, spleen and kidney while brain showed little change. The inhibitor did not act on glucuronidase in vitro, so the effect of the inhibitor is complex, possibly a result of the loss of glucosidase activity. Since glucuronidase contains glucose, we suggest that the transport of the enzyme between subcellular regions and tissues involves loss of part of the glucose moieties. 2. Levels of glucocerebrosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) dropped very rapidly after epoxide injection, reaching a minimum at 1 h in liver. There was a noticeable restoration of activity within the next 1--2 h. Aryl beta-glucosidase (EC 3.2.1.21) decrease somewhat less than cerebrosidase, reaching a minimum within 2 h. It too showed some recovery of activity within 3 h. 3. Acid phosphatase rose slightly in liver but not in brain. alpha-L-Fucosidase and angiotensin-converting enzyme were not affected by the epoxide injection. The latter two enzymes are known to contain glucose. 4. Injection of a hemolyzing agent, phenylhydrazine, produced an increased level of glucuronidase in liver and spleen within 6 days, but not in kidney. This enhancement was a little less in mice previously injected with the glucosidase inhibitor. 5. Mice injected with the epoxide once a day eight times showed a distinct rise in brain glucuronidase level, as well as a rise in brain weight. However, the other organs showed only the same decrease in glucuronidase specific activity noted with the single injection protocol. It is suggested that the difference is due to the blood-brain barrier, which could slow the loss of brain glucuronidase from the extracellular fluid.     

Purification and characterization of leukotriene A4 epoxide hydrolase from dog lung

Leukotriene A4 epoxide hydrolase from dog lung, a soluble enzyme catalyzing the hydrolysis of leukotriene A4 (LTA4) to leukotriene B4 (LTB4) was partially purified by anion exchange HPLC. The enzymatic reaction obeys Michaelis- Menten kinetics. The apparent Km ranged between 15 and 25 microM and the enzyme exhibited an optimum activity at pH 7.8. An improved assay for the epoxide hydrolase has been developed using bovine serum albumin and EDTA to increase the conversion of LTA4 to LTB4. This method was used to produce 700 mg of LTB4 from LTA4 methyl ester. The partial by purified enzyme was found to be uncompetitively inhibited by divalent cations. Ca+2, Mn+2, Fe+2, Zn+2 and Cu+2 were found to have inhibitor constants (Ki) of 89 mM, 3.4 mM, 1.1 mM, 0.57 mM, and 28 microM respectively Eicosapentaenoic acid was shown to be a competitive inhibitor of this enzyme with a Ki of 200 microM. From these inhibition studies, it can be theorized that the epoxide hydrolase has at least one hydrophobic and one hydrophilic binding site.     

Active site directed inhibition of a cytosolic beta-glucosidase from calf liver by bromoconduritol B epoxide and bromoconduritol F

Hydrolysis of p-nitrophenyl-beta-D-glucoside by cytosolic beta-glucosidase proceeds with retention of the anomeric configuration. Whereas inactivation of the enzyme by the glucosidase inhibitor conduritol B epoxide (CBE) was extremely slow (ki(max)/Ki 0.57 M-1 min-1) it reacted 130 times more rapidly with 6-bromo-6-deoxy-CBE (Br-CBE). The beta-glucosidase could be labeled with [3H]Br-CBE; incorporation of 1 mol inhibitor/mol enzyme resulted in complete loss of activity. Most of the bound inhibitor was released after denaturation and treatment with ammonia as (1,3,4/2,5,6)-6-bromocyclohexanepentol, thus demonstrating the formation of an ester bond with an active site carboxylate by trans-diaxial opening of the epoxide ring. It was concluded from the Ki values for the epoxide inhibitors and for coduritol B with the cytosolic enzyme and corresponding data for the lysosomal beta-glucosidase that the unusually low reactivity with CBE and Br-CBE is probably due to the inability of the cytosolic enzyme to effectively donate a proton to the epoxide oxygen. An extremely rapid inactivation of the cytosolic beta-glucosidase was caused by bromoconduritol F ((1,2,4/3)-1-bromo-2,3,4-trihydroxycyclohex-5-ene) with ki(max)/Ki 10(5) M-1 min-1. In contrast with the Br-CBE-inhibited enzyme the beta-glucosidase inhibited by bromoconduritol F was subject to spontaneous reactivation with t1/2 approximately 20 min.     

Distribution of conduritol B epoxide in the animal model for Gaucher's disease (Gaucher mouse)

The time course of the distribution of the beta-glucosidase inhibitor [3H]conduritol B epoxide was determined in various organs of mice, which had received a single interperitoneal dose of the inhibitor. The epoxide is rapidly distributed over all tissues except brain where its concentration is only one-tenth of the average. This is considered an indication that the epoxide can pass the blood/brain barrier only with difficulty. A 4-fold enrichment is seen in the kidney. The inhibitor is excreted with a half-life of about 7 h; it is not metabolized. A parallel determination of beta-glucosidase activity in the tissues showed greater than 90% inhibition within 1 and 2 h and a beginning recovery between 4 and 12 h. The only exception was brain, where no effects could be seen after 1 h and where a subsequent decrease to 37% of normal was observed after 12 h.     

Inactivation of alpha-glucosidase by the active-site-directed inhibitor, conduritol B epoxide

Conduritol B epoxide is an active-site-directed inhibitor of some glucosidases. The inactivation of alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from Monascus ruber by conduritol B epoxide is irreversible and first-order with respect to time and inhibitor concentration. The inactivation is prevented by the presence of the substrate maltose. The pH-dependence of Vmax for maltose indicated the participation of two dissociating groups with pK values of 4.1 and 5.8 in the enzyme-substrate complex. Modification of the alpha-glucosidase with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride led to loss of activity, which suggests that a carboxyl group(s) is located at the active site of alpha-glucosidase.     

Isolation and characterization of N98-1272 A, B and C, selective acetylcholinesterase inhibitors from metabolites of an actinomycete strain

A high throughput screening was carried out in order to search for inhibitors of acetylcholinesterase (AChE) from microorganism metabolites. An actinomycete strain was found to produce active compounds named N98-1272 A, B and C with IC50 of 15.0, 11.5, 12.5 microM, respectively. Structural studies revealed that the three compounds are identical to the known antibiotics, Manumycin C, B and A. Kinetic analyses showed that N98-1272 C (Manumycin A) acted as a reversible noncompetitive inhibitor of acetylcholinesterase, with a Ki value of 7.2 microM. The cyclohexenone epoxide part of the structure plays a crucial role in the inhibitory activity against AChE. Compared with Tacrine, N98-1272 A, B, and C exhibit much better selectivity toward AChE over BuChE.     

A sensitive and reproducible fluorescent-based HPLC assay to measure the activity of acid as well as neutral beta-glucocerebrosidases

The activity of lysosomal acid β-glucocerebrosidase (AGC, EC 3.2.1.45), which hydrolyzes the O-glycosidic linkage between d-glucose and ceramide of glucosylceramide (GlcCer), is a marker for the diagnosis of Gaucher disease because the disease is caused by dysfunction of AGC due to mutations in the gene. The activity of AGC is potently inhibited by conduritol B epoxide (CBE), whereas CBE-insensitive nonlysosomal neutral β-glucocerebrosidase (NGC) activities have been found in various vertebrates, including humans. We report here a new reliable method to determine AGC as well as NGC activities using normal-phase high-performance liquid chromatography (HPLC) and NBD (4-nitrobenzo-2-oxa-1,3-diazole)- or BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-labeled GlcCer as a substrate. The reaction products of the enzymes, C6-NBD-ceramide and C12-BODIPY-ceramide, were clearly separated from the corresponding substrates on a normal-phase column within 5 min using a different solvent system. Reaction products could be detected quantitatively at concentrations ranging from 50 fmol to 50 pmol for C6-NBD-ceramide and from 10 fmol to 5 pmol for C12-BODIPY-ceramide. V_max/K_m values of human fibroblast AGC for fluorescent GlcCer were much higher than those for 4-methylumbelliferyl-β-d-glucoside (4MU-Glc), which is used prevalently for Gaucher disease diagnosis. As a result, AGC activity was detected quantitatively using fluorescent GlcCer, but not 4MU-Glc, using 5 μl of human serum or 1 × 10⁴ cultured human fibroblasts. The current method clearly showed the decrease of AGC activities in fibroblasts and serum from the patient with Gaucher disease compared with normal individuals, suggesting that the method is applicable for the diagnosis of Gaucher disease. Furthermore, this method was found to be useful for measuring the activities of nonlysosomal NGC of various cells and tissues in the presence of CBE.     

Isofagomine increases lysosomal delivery of exogenous glucocerebrosidase

Intravenous enzyme replacement therapy (ERT) with purified glucocerebrosidase (GLA) leads to significant improvement of the clinical manifestations in patients with Type 1 Gaucher disease. However, the high doses required, slow response and inability to recover most of the infused enzyme in the target tissues may be attributed to losses occurring during transit en route to the lysosome. Preincubation of GLA with isofagomine (IFG), a slow-binding inhibitor, significantly increased stability of the enzyme to heat, neutral pH and denaturing agents in vitro. Preincubation of GLA with isofagomine prior to uptake by cultured cells results in increased intracellular enzyme activity accompanied by an increase in enzyme protein suggesting that reduced denaturation of GLA in the presence of isofagomine leads to a decrease in the degradation of the enzyme after internalization. Preincubation of GLA with slow-binding inhibitors before infusion may improve the effectiveness of ERT for Gaucher disease.     

Hydrophilic iminosugar active-site-specific chaperones increase residual glucocerebrosidase activity in fibroblasts from Gaucher patients

Gaucher disease is an autosomal recessive lysosomal storage disorder caused by the deficient activity of glucocerebrosidase. Accumulation of glucosylceramide, primarily in the lysosomes of cells of the reticuloendothelial system, leads to hepatosplenomegaly, anemia and skeletal lesions in type I disease, and neurologic manifestations in types II and III disease. We report herein the identification of hydrophilic active-site-specific chaperones that are capable of increasing glucocerebrosidase activity in the cultured fibroblasts of Gaucher patients. Screening of a variety of natural and synthetic alkaloid compounds showed isofagomine, N-dodecyl deoxynojirimycin, calystegines A3, B1, B2 and C1, and 1,5-dideoxy-1,5-iminoxylitol to be potent inhibitors of glucocerebrosidase. Among them, isofagomine was the most potent inhibitor of glucocerebrosidase in vitro, and the most effective active-site-specific chaperone capable of increasing residual glucocerebrosidase activity in fibroblasts established from Gaucher patients with the most prevalent Gaucher disease-causing mutation (N370S). Intracellular enzyme activity increased approximately two-fold after cells had been incubated with isofagomine, and the increase in glucocerebrosidase activity was both dose-dependent and time-dependent. Western blotting demonstrated that there was a substantial increase in glucocerebrosidase protein in cells after isofagomine treatment. Immunocytochemistry revealed an improvement in the glucocerebrosidase trafficking pattern, which overlaps that of lysosome-associated membrane protein 2 in Gaucher fibroblasts cultivated with isofagomine, suggesting that the transport of mutant glucocerebrosidase is at least partially improved in the presence of isofagomine. The hydrophilic active-site-specific chaperones are less toxic to cultured cells. These results indicate that these hydrophilic small molecules are suitable candidates for further drug development for the treatment of Gaucher disease.     

Analyses of variant acid beta-glucosidases: effects of Gaucher disease mutations

Acid beta-glucosidase (GCase) is a 497-amino acid, membrane-associated lysosomal exo-beta-glucosidase whose defective activity leads to the Gaucher disease phenotypes. To move toward a structure/function map for disease mutations, 52 selected single amino acid substitutions were introduced into GCase, expressed in an insect cell system, purified, and characterized for basic kinetic, stability, and activator response properties. The variant GCases from Gaucher disease patients and selected variant GCases from the mouse had decreased relative k(cat) and differential effects on active site binding and/or attachment of mechanism-based covalent (conduritol B epoxide) or reversible (deoxynojirimycin derivatives) inhibitors. A defect in negatively charged phospholipid activation was present in the majority of variant GCases but was increased in two, N370S and V394L. Deficits in saposin C enhancement of k(cat) were present in variant GCases involving residues 48-122, whereas approximately 2-fold increases were obtained with the L264I GCase. About 50% of variant GCases each had wild-type or increased sensitivity to in vitro cathepsin D digestion. Mapping of these properties onto the crystal structures of GCase indicated wide dispersion of functional properties that can affect catalytic function and stability. Site-directed mutagenesis of cysteine residues showed that the disulfide bonds, Cys(4)-Cys(16) and Cys(18)-Cys(23), and a free Cys(342) were essential for activity; the free Cys(126) and Cys(248) were not. Relative k(cat) was highly sensitive to a His substitution at Arg(496) but not at Arg(495). These studies and high phylogenetic conservation indicate localized and general structural effects of Gaucher disease mutations that were not obvious from the nature of the amino acid substitution, including those predicted to be nondisruptive (e.g. Val --> Leu). These results provide initial studies for the engineering of variant GCases and, potentially, molecular chaperones for therapeutic use.     

Enzyme replacement therapy: conception,chaos and culmination

Soon after the enzymatic defects in Gaucher disease and in Niemann-Pick disease were discovered, enzyme replacement or enzyme supplementation was proposed as specific treatment for patients with these and related metabolic storage disorders. While relatively straightforward in concept, successful implementation of this approach required many years of intensive effort to bring it to fruition. Procedures were eventually developed to produce sufficient quantities of the requisite enzymes for clinical trials and to target therapeutic enzymes to lipid-storing cells. These achievements led to the development of effective enzyme replacement therapy for patients with Gaucher disease and for Fabry disease. These demonstrations provide strong incentive for the application of this strategy for the treatment of many human disorders of metabolism.     

Characterization of 2-bromoethanesulfonate as a selective inhibitor of the coenzyme m-dependent pathway and enzymes of bacterial aliphatic epoxide metabolism

Bacterial growth with short-chain aliphatic alkenes requires coenzyme M (CoM) (2-mercaptoethanesulfonic acid), which serves as the nucleophile for activation and conversion of epoxide products formed from alkene oxidation to central metabolites. In the present work the CoM analog 2-bromoethanesulfonate (BES) was shown to be a specific inhibitor of propylene-dependent growth of and epoxypropane metabolism by Xanthobacter autotrophicus strain Py2. BES (at low [millimolar] concentrations) completely prevented growth with propylene but had no effect on growth with acetone or n-propanol. Propylene consumption by cells was largely unaffected by the presence of BES, but epoxypropane accumulated in the medium in a time-dependent fashion with BES present. The addition of BES to cells resulted in time-dependent loss of epoxypropane degradation activity that was restored upon removal of BES and addition of CoM. Exposure of cells to BES resulted in a loss of epoxypropane-dependent CO(2) fixation activity that was restored only upon synthesis of new protein. Addition of BES to cell extracts resulted in an irreversible loss of epoxide carboxylase activity that was restored by addition of purified 2-ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of epoxide carboxylation, but not by addition of epoxyalkane:CoM transferase or 2-hydroxypropyl-CoM dehydrogenase, the enzymes which catalyze the first two reactions of epoxide carboxylation. Comparative studies of the propylene-oxidizing actinomycete Rhodococcus rhodochrous strain B276 showed that BES is an inhibitor of propylene-dependent growth in this organism as well but is not an inhibitor of CoM-independent growth with propane. These results suggest that BES inhibits propylene-dependent growth and epoxide metabolism via irreversible inactivation of the key CO(2)-fixing enzyme 2-KPCC.