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PubMed2004

Structure-activity relationships for alpha-glucosidase inhibition of baicalein, 5,6,7-trihydroxyflavone: the effect of A-ring substitution.

In order to estimate the effects of the A-ring hydroxyl group of baicalein (5,6,7-trihydroxyflavone, 1) on rat intestinal alpha-glucosidase inhibition, flavone, monohydroxyflavones, dihydroxyflavones, and methylated derivatives of 5,6,7-trihydroxyflavone were used for the structure-activity relationship (SAR) study. The importance of the 6-hydroxyl group of baicalein was validated for an exertion of the activity. And also, the tested flavones which lacked a hydroxyl substituent on any of positions 5, 6, or 7, showed no activity. Hence, the 5,6,7-trihydroxyflavone structure was concluded to be crucial for the potent inhibitory activity. In addition, an introduction of electron-withdrawing or electron-donating groups at position 8 of baicalein led to a dramatic decrease for activity, except for 8-fluoro-5,6,7-trihydroxyflavone, which carried a less bulky substituent on position 8. Hence, this result suggested that a sterically bulky substituent on C-8 of baicalein was detrimental for the activity regardless of its electronic nature. Through examining the inhibitory mechanism of baicalein against rat intestinal alpha-glucosidase, it was suggested to be a mixed type inhibition.

Animals↗
PubMed2003

Production, purification, and biochemical characterization of two beta-glucosidases from Sclerotinia sclerotiorum.

The filamentous fungus Sclerotinia sclerotiorum produces beta-glucosidases in liquid culture with a variety of carbon sources, including cellulose (filter paper), xylan, barley straw, oat meal, and xylose. Analysis by native polyacrylamide gel electrophoresis (PAGE) followed by an activity staining with the specific chromogenic substrate, 5-bromo 4-chloro 3-indolyl beta-1,4 glucoside (X-glu) showed that two extracellular beta-glucosidases, designated as beta-glu1 and beta-glu2, were in the filter paper culture filtrate. Only one enzyme designated as beta-glu x was revealed by the same method in the xylose culture filtrate. Beta-glu1 and beta-glu2 were purified to homogeneity. The purification procedure consist of a common step of anion-exchange chromatography on DEAE-Sepharose CL6B, both high-performance liquid chromatography (HPLC) anion-exchange and gel filtration columns for beta-glu1 and only HPLC gel filtration for beta-glu2. Beta-glu1 has a molecular mass of 196 kDa and 96.5 kDa, as estimated by gel filtration and sodium dodecyl sulfate (SDS)-PAGE, respectively, suggesting that the native enzyme may consist of two identical subunits. The same analysis showed that beta-glu2 is a monomeric protein with an apparent molecular mass of about 76.5 kDa. Beta-glu1 and beta-glu2 hydrolyses PNPGlc and cellobiose, with apparent Km values respectively for PNPGlc and cellobiose of 0.1 and 1.9 mM for beta-glu1 and 2.8 and 8 mM for beta-glu2. Both enzymes exhibit the same temperature and pH optima for PNPGlc hydrolysis (60 degrees C and pH 5.0). beta-glu1 was stable over a pH range of 3-8 and kept 50% of its activity after 30 min of heating at 60 degrees C without substrate. It was further characterized by studying the effect of some cations and various reagents on its activity.

Ascomycota↗
PubMed1999

Miglitol, a new alpha-glucosidase inhibitor.

Miglitol (Bay m 1099, Bayer) is a second generation alpha-glucosidase inhibitor. It is a derivative of 1-desoxynojirimycin, and binds reversibly to the brushborder alpha-glucosidase enzymes. In contrast to its parent drug (acarbose, Bay g 5421, Bayer), miglitol is almost completely absorbed in the small intestine. It has to be taken with each main meal, and through its effect on carbohydrate digestion it blunts the postprandial blood glucose increase. Miglitol has no or a very small effect on fasting blood glucose levels. The blood-glucose lowering effects of miglitol in patients with Type 2 diabetes are lower than those of the frequently-used sulphonylurea compounds. Long-term studies show that a moderate average reduction of HbA1c of 0.3-0.7% point from baseline can be achieved. An advantage over sulphonylurea is the effect on serum insulin levels: miglitol therapy leads to slightly lower postprandial levels of serum insulin, whereas chronic sulphonylurea treatment usually increases serum insulin levels. This insulin-sparing effect may, in theory, lead to a lesser weight gain or even no weight gain and reduced risk of hypoglycaemia during chronic treatment. Long-term experience in Type 1 diabetic patients is limited. Similarly, miglitol may lead to reduced postprandial glucose excursions, slightly reduced insulin requirements and perhaps, as a consequence, a lower risk of hypoglycaemia. More long-term data are needed to fully assess to the clinical use of miglitol in these patients.

1-Deoxynojirimycin↗
PubMed1992

Oral antidiabetic agents. The emergence of alpha-glucosidase inhibitors.

Hyperglycaemia in patients with non-insulin-dependent diabetes mellitus (NIDDM) results from impaired insulin action and/or deficient insulin secretion. These abnormalities lead to increased hepatic glucose production, the primary cause of fasting hyperglycaemia, and decreased peripheral glucose uptake, the major mechanism responsible for postprandial hyperglycaemia. Hyperglycaemia in patients with NIDDM can be decreased by several different mechanisms: (1) a decrease in nutrient ingestion; (2) an increase in insulin secretion; (3) a decrease in hepatic glucose production; (4) an increase in peripheral glucose uptake. Oral agents used to treat NIDDM operate through 1 or more of the above mechanisms. alpha-Glucosidase inhibitors, a new class of drugs that delay carbohydrate digestion and absorption, reduce postprandial glycaemic rises by about 3 mmol/L. Metformin decreases fasting and postprandial hyperglycaemia through increasing glucose uptake and perhaps decreasing appetite. Sulphonylureas lower hyperglycaemia by increasing insulin secretion and to a lesser degree potentiating insulin action on the liver and peripheral tissues. alpha-Glucosidase inhibitors are particularly useful as primary therapy for patients with mild to moderate hyperglycaemia and in those patients who may be at risk for hypoglycaemia or lactic acidosis. Sulphonylureas are indicated for the more severely hyperglycaemic NIDDM patients who are not yet candidates for insulin therapy. Metformin is useful in obese moderately hyperglycaemic NIDDM patients. These oral agents can be used in combination to give better glycaemic control than is possible with each alone.

Administration, Oral↗
PubMed2002

Identification of novel alpha-glucosidase inhibitors by screening libraries based on N- [4-(benzyloxy) benzoyl] alanine derivatives.

A library of 72 compounds related to N- [4-(benzyloxy) benzoyl]alanine (I) was synthesized, prepared and screened for alpha-glucosidase inhibitory activity. Four compounds showed potent inhibition, six compounds moderate inhibition, and 16 were weak inhibitors. One compound, N- [4-(benzyloxy) benzoyl] serine, was found to be a potent inhibitor of alpha-glucosidase with 100% inhibition at 1 micro M. This inhibitor was at least five times more potent than the lead compound I.

Alanine↗
PubMed1996

Does suppression of postprandial blood glucose excursions by the alpha-glucosidase inhibitor miglitol improve insulin sensitivity in diet-treated type II diabetic patients?

OBJECTIVE: Insulin sensitivity is impaired in patients with type II diabetes and is exacerbated by high mean blood glucose (BG). Potentially, large postprandial swings in BG could result in further decrements of insulin sensitivity. Because alpha-glucosidase inhibitors cause a marked reduction in the amplitude of BG changes, the aim of this study was to determine if such a BG-smoothing effect improves insulin sensitivity in well-controlled type II diabetic subjects treated with diet alone. RESEARCH DESIGN AND METHODS: Patients received either miglitol (BAY m 1099) (50 mg three times daily) or placebo for 8 weeks in a randomized double-blind parallel study. The miglitol (9 men, 2 women) and placebo (7 men, 3 women) groups were well matched (mean +/- SD) for age, weight, and blood glucose control (fasting BG, 6.4 +/- 1.0 vs. 6.9 +/- 1.6 mmol/l; HbA1, 7.7 +/- 1.0 vs. 7.9 +/- 0.4%; fructosamine, 0.99 +/- 0.08 vs. 1.07 +/- 0.17 mmol/l). The glucose metabolic clearance rate was calculated during the last 30 min of a 150 min glucose/insulin sensitivity test (glucose, 6 mg . kg-1 . min-1; insulin, 0.5 U . kg-1 . min-1). RESULTS: There was no significant improvement in metabolic clearance rate (0.21 +/- 0.27 vs. 0.16 +/- 0.35 l . kg-1 . min-1) for the miglitol- and placebo-treated groups, respectively. There were no statistically significant differences between miglitol and placebo for changes from baseline in BG (0.1 +/- 0.1 vs. -0.1 +/- 0.2 mmol/l), HbA1 (0.1 +/- 0.1 vs. 0.3 +/- 0.1%), and fructosamine (-0.06 +/- 0.02 vs. -0.03 +/- 0.02 mmol/l). CONCLUSIONS: Alpha-glucosidase-induced improvement in postprandial hyperglycemia does not result in increased insulin sensitivity.

1-Deoxynojirimycin↗
PubMed1998

Chronic treatment of African-American type 2 diabetic patients with alpha-glucosidase inhibition.

OBJECTIVE: To evaluate the long-term efficacy, safety, and tolerability of the alpha-glucosidase inhibitor miglitol in the treatment of African-American patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: A total of 345 African-American type 2 diabetic patients (mean age 55.6 years, BMI 31.9 kg/m2, duration of diabetes 4.9 years, baseline HbA1C 8.7%) treated with either diet alone or sulfonylurea were randomized to 1 year of double-blind treatment with either placebo (n = 117) or miglitol (n = 228) at doses of 50 or 100 mg t.i.d., titrated based on tolerability. The primary efficacy criterion was change from baseline in HbA1C at the 6-month visit. Secondarily efficacy parameters included changes from baseline in plasma glucose and serum insulin (both fasting and 120 min after a standardized test meal), fasting lipids, and urinary albumin-to-creatinine ratio. Safety and tolerability evaluations were primarily based on reporting of adverse events and symptoms and on periodic laboratory analyses. RESULTS: Miglitol treatment was associated with a mean placebo-subtracted reduction in HbA1C from baseline of 1.19% at 6 months. Fasting and 120-min postprandial plasma glucose levels were reduced in parallel to HbA1C, in association with miglitol treatment. Significant reductions versus placebo in 120-min postprandial insulin levels, in LDL cholesterol, and in fasting triglycerides, were also seen in the miglitol group at individual study time points. Softer, more frequent stools and flatulence were significantly more common in the miglitol group. Urinary tract infections, hematuria, and herpes simplex infections were significantly more common in the placebo group. CONCLUSIONS: Miglitol treatment appears to be at least as efficacious in the African-American type 2 population as in the U.S. type 2 population at large, with comparable tolerability. alpha-Glucosidase treatment may be an important therapeutic option in these patients in view of their greater risk for microvascular complications and the accumulating body of evidence that better glucose control reduces the risk of these complications.

1-Deoxynojirimycin↗
PubMedDate not supplied

A new approach to the treatment of nocturnal hypoglycemia using alpha-glucosidase inhibition.

Nocturnal hypoglycemia is common in the diabetic patient on twice-daily regular and intermediate (NPH or lente) insulin regimens because intermediate-acting insulins before the main evening meal produce "unopposed" free insulin peaks around 0300 h, food absorption having been completed much earlier. Fourteen insulin-dependent diabetic patients were treated for 6 wk with the alpha-glucosidase inhibitor, acarbose, in a double-blind crossover study to see whether the drug would delay absorption of the evening meal sufficiently to correct the mismatch and prevent nocturnal hypoglycemia. On 200 mg acarbose (six patients), inhibition of carbohydrate digestion was so profound as to lead to midevening hypoglycemia with severe flatulence and abdominal colic. With a smaller dose of 100 mg before the evening meal (eight patients) there was a significant reduction in MAGE and MBG coupled with a clinically significant reduction in midevening and nocturnal hypoglycemic reactions. Alpha-glucosidase inhibition therefore provides a promising new approach to the problem of nocturnal hypoglycemia although a preparation that is safe for long-term clinical use remains to be found.

Acarbose↗
PubMed2002

Novel alpha-glucosidase inhibitors, CKD-711 and CKD-711a produced by Streptomyces sp. CK-4416. I. Taxonomy, fermentation and isolation.

New alpha-glucosidase inhibitors, CKD-711 and CKD-711a were produced from the fermentation broth of Streptomyces sp. CK-4416 which was isolated from a forest soil of Jeju Island, South Korea. CKD-711 and CKD-711a were purified by Dowex 50W-2X and Sephadex G-10 column chromatography. In in vitro studies, CKD-711 showed a potent inhibitory activity against a-glucosidase from mammalian, but less inhibition against a-amylase from microorganism and mammalian. CKD-711a showed a lower inhibitory activity than CKD-711.

Animals↗
PubMed2002

Dose-dependent efficacy of miglitol, an alpha-glucosidase inhibitor, in type 2 diabetic patients on diet alone: results of a 24-week double-blind placebo-controlled study.

AIMS: A double-blind randomised study was performed to compare the dose-effect and dose-tolerability relationships between the alpha-glucosidase inhibitor miglitol in doses of 25 mg, 50 mg, 100 mg and 200 mg all t.i.d. vs placebo t.i.d. in patients with Type 2 diabetes mellitus on diet only. METHODS: After a 6-week placebo run-in period 468 patients with a fasting blood glucose > or = 7 mmol/l as well as a HbA1c between 6.1% and 10.4% were randomised for a 24-week treatment period. RESULTS: The results of 465 patients were valid for safety analysis and of 384 patients for the efficacy analysis. In the placebo group the HbA1c level increased by 0.40+/-1.46% as compared with baseline. The decrease in the mean HbA1c values (corrected for differences in baseline values) was significant and dose-dependent for all miglitol groups compared with placebo, being -0.46% (95% CI: -0.91%, -0.01%) in the 25 mg group, -0.45% (95% CI: -0.90%, -0.003%) in the 50 mg group, -0.84% (95% CI: -1.31%, -0.37%) in the 100 mg group and -1.26% (95% CI: -1.76%, -0.76%) in the 200 mg group. Blood glucose levels following a standardised breakfast tolerance test were significantly and dose-dependently lower for all the miglitol doses at 12 and 24 wk of treatment compared to baseline: in comparison with baseline maximum blood glucose increased by 4% with placebo and decreased by 7%, 14%, 24% and 33% with miglitol 25 mg, 50 mg, 100 mg and 200 mg t.i.d. respectively. The same pattern was seen with postprandial maximal serum insulin levels which decreased by 8% under placebo and by 17%, 26%, 25% and 35% with the 25 mg to 200 mg doses of miglitol. The adverse events reported were mainly of gastrointestinal nature, mostly being flatulence, diarrhoea and abdominal pain and the incidence increased with increasing dose. Although the side effects were not serious, they were troublesome, leading to a considerable drop-out rate increasing with dose. CONCLUSIONS: The alpha-glucosidase inhibitor miglitol in Type 2 diabetic patients on diet alone decreases both HbA1c levels and postprandial glucose and insulin levels in a dose-dependent manner. Gastrointestinal side effects also showed dose-dependency. Combination of efficacy and safety results leads to the conclusion that the optimal dose of miglitol will be in the range of 50 to 100 mg t.i.d.

1-Deoxynojirimycin↗
PubMed1982

Correlation between L-Carnitine and alpha-1, 4-glucosidase activity in the semen of normal, infertile and vasectomized men.

The semen content of L-carnitine and of alpha 1, 4-glucosidase has been measured in subjects consulting for evaluation of their fertility. A close correlation (r=0.684) was found between both parameters over the range of azoospermia to normal zoospermia. A significant number of patients with oligo or azoospermia displayed normal values of L-carnitine and of alpha-1, 4-glucosidase while approximately 50% showed levels in the low spectrum of vasectomized men. On the basis of these findings, an obstructive pathology at epididymal or vas deferens level was established by vasography and/or bilateral scrotal exploration in 9 patients with azoospermia. These 2 epididymal markers might thus be useful in the hands of the practicing andrologist who has to determine precisely the site of a dysfunction in the reproductive system which leads to infertility.

Biology↗
PubMed1992

[Alpha-glucosidase inhibitors of microbial origin].

The data on spreading of inhibitors of alpha-glucosidases with microbial origin are given. Physiochemical characteristics of acorbose--a known inhibitor of alpha-glucosidases--and new inhibitor isolated from Streptomyces sp. are given in detail.

Acarbose↗
PubMed2000

[Treatment of type 2 diabetes mellitus with the alpha-glucosidase inhibitor Miglitol in the doctor's office].

BACKGROUND AND METHOD: In an observational study carried out in accordance with paragraph 67 (6) of the AMG and beginning in May 1998, 846 patients with type 2 diabetes mellitus were followed for a period of three months by general practitioners and internists. The aim was to acquire data on the efficacy and tolerability of the alpha-glucosidase inhibitor, Miglitol, used in the doctor's office setting. A major point of interest was to determine the feasibility of using a regimen of gradually increasing doses in daily practice. The parameters investigated were HbA1c, fasting blood glucose and changes in the patient's weight. Any side effects were recorded by the care-providing physician and evaluated in the global tolerability analysis. RESULTS: The demographic data showed a balanced distribution of male and female patients. The average age of the men was 62 years, of the women 65 years. At the start of the observation, 62% of the patients had polyneuropathy, 35% retinopathy, and 25% nephropathy. 620 patients (73.3%) had received prior antidiabetic treatment. Depending on tolerability, the dose of Miglitol was increased individually from an initial 3 x 50 mg to 3 x 100 mg. 63.8% of the patients remained at the initial dose of 3 x 50 mg. A clear decrease in both HbA1c and fasting blood glucose was observed under treatment. During the course of the three months, the BMI decreased slightly by an average of 0.4 kg/m2, a desirable side effect in this patient group. Adverse reactions occurred in 39 ofthe 846 patients in the form of flatulence, diarrhea and enterospasm. CONCLUSION: Treatment with the alpha-glucosidase inhibitor Miglitol proved to be highly effective and readily combinable with other antidiabetics. Its efficacy was assessed to be "very good" or "good" by 86.5% of the physicians, and more than 90% assessed its tolerability to be "very good" or "good".

1-Deoxynojirimycin↗
PubMed2001

[The alpha-glucosidase inhibitor miglitol for the treatment of type 2 diabetes mellitus in the doctor's office].

BACKGROUND AND METHOD: An observational study was carried out according to and 67 (6) AMG from october 1999 until august 2000. 2654 patients with diabetes mellitus type 2 were treated by 766 physicians for 3 months with alpha-glucosidase inhibitor miglitol. The aim of the study "DiaLife" was to obtain data regarding the effectiveness and safety of miglitol in the daily practice. Pre- and postprandial glucose levels, HbA1c, triglycerides, urin glucose, microalbuminuria and body weight were used as parameters of effectiveness. Also an increasing treatment scheme was used. Side effects were documented by the care providing physicians and evaluated by the global tolerability analysis. RESULTS: Males and females showed a balanced distribution. The average age was 62 years and the average duration of diabetes was 5 years. Concomitant diseases were described for 82% of patients: 62% of patients suffered from hypertension, 43% from hyperlipidemia and 22% from coronary heart disease. 64% of patients were already on treatment for diabetes. The mostly prescribed drugs were sulfonylureas and biguanides. Miglitol was gradually increased (first week 1 x 50 mg daily, second week 2 x 50 mg daily, until the third week 3 x 50 mg daily) and the final dosages were 3 x 50 mg or 3 x 100 mg miglitol daily. HbA1c, pre- and postprandial glucose levels were strongly reduced. The HbA1c decreased from 8.4% to 7,1%. Pre- and postprandial glucose levels were reduced minus 46 mg/dl and minus 59 mg/dl. The average decrease of body weight was 1,9 kg; the effect was augmented in individuals with higher BMI. Adverse effects were reported in 47 patients and occured mainly in the GI tract. There were no problems in the combination of miglitol with other pharmacological substance groups. CONCLUSION: The alpha-glucosidase inhibitor miglitol is an efficient and safe drug for the treatment of diabetes mellitus type 2. It is an ideal tool for mono- and combination therapy. It reduces remarkably the body weight. 90% of physicians judged miglitol's efficiency and safety as "very good" or "good".

1-Deoxynojirimycin↗
PubMed1991

Post-prandial glycaemic reduction by an alpha-glucosidase inhibitor in type 2 diabetic patients with therapeutically attained basal normoglycaemia.

Post-prandial glucose excursions remain elevated in most patients with diabetes even when normal fasting plasma glucose levels have been achieved. In 39 patients with type 2 diabetes who had attained basal normoglycaemia by therapy with diet alone, a sulphonylurea, a basal insulin supplement or basal plus prandial insulin the mean glycosylated haemoglobin (HbA1) values were at the upper end (mean +/- 1SD, 8.1 +/- 1.1%) of the normal range (5.0-8.2%). Miglitol, an alpha-glucosidase inhibitor, given in a dose of 50 mg three times a day was studied in a double blind randomized crossover study. In diet and sulphonylurea treated patients, a mean 25% reduction of the post-prandial plasma glucose excursions was obtained whereas in ultralente treated patients miglitol appeared to reduce basal plasma glucose levels (p < 0.006). Side effects were limited to minor gastrointestinal disturbances, usually ameliorating after the first week of therapy. Alpha-glucosidase inhibition to prevent post-prandial glycaemia may have a role in patients in whom sulphonylurea or diet therapy has been used to obtain normal basal glucose concentrations.

1-Deoxynojirimycin↗
PubMed1989

Metabolic improvements associated with a reduction of abdominal visceral fat caused by a new alpha-glucosidase inhibitor, AO-128, in Zucker fatty rats.

The relationship between mesenteric fat accumulation and metabolic disorder was studied in genetically obese Zucker fatty rats. These animals had high levels of plasma glucose, triglyceride and total cholesterol as well as increased liver triglyceride in comparison with lean rats. In addition, portal free fatty acid (FFA) levels were also higher in fatty rats than in lean rats. Direct measurement of fat weight revealed a substantial increase of mesenteric as well as subcutaneous fat. The volume of mesenteric fat cells was greater than that of subcutaneous fat cells in Zucker fatty rats (1.67 +/- 0.49 nl versus 1.00 +/- 0.31 nl), although the mesenteric fat cell volume was less than half of the subcutaneous fat cell volume (0.05 +/- 0.02 nl versus 0.14 +/- 0.07 nl) in lean rats. An increase in mesenteric fat cell volume was thus more predominant than that of subcutaneous fat cells in the fatty rats. Administration of AO-128 (50 p.p.m./day), a new alpha-glucosidase inhibitor, caused a substantial reduction of mesenteric fat weight accompanied by a marked decrease in fat cell volume in Zucker fatty rats. The drug also caused a significant reduction of FFA levels in the portal vein in parallel with a marked reduction of triglyceride content in the liver. These observations indicate that the fat accumulation in the markedly enlarged mesenteric fat cells is related to the elevated levels of FFA in the portal vein, which in turn may cause metabolic disorders in the liver. A new alpha-glucosidase inhibitor improves these disorders, at least in part, by preventing an enlargement of mesenteric fat cells.

Adipose Tissue↗
PubMed1980

[Glycogen storage disease by amylo 1,6-glucosidase deficiency (author's transl)].

A case of liver glycogen storage disease with amylo 1,6-glucosidase deficiency is reported. Enlarged liver was found at birth, and it is now accompanied by splenomegaly, low fasting blood glucose with ketonuria, elevation of transaminase values and glycogen accumulation with connective periportal tissue in liver histological study. In this glucogenosis results of functional tests on carbohidrate metabolism and glycogen enzymatic assay showed a direct relationship between functional and biochemical behaviour of liver cells. Amylo 1,6-glucosidase deficiency is accompanied by absence of glucogenolysis when glucagon is administrated after a long fast, and an increase of blood glucose when glucagon is administrated after food ingestion. Glycolisis tests show blood lactate elevation when some hexose or alanine are administrated; glyconeogenesis tests show blood glucose elevation when hexose, alanine or glycerol are administrated.

Glycogen↗
PubMed1994

Analysis of human acid beta-glucosidase by site-directed mutagenesis and heterologous expression.

Structure/function relationships of acid beta-glucosidase, the enzyme deficient in Gaucher disease, were evaluated by characterizing the proteins expressed from cDNAs encoding normal and mutant enzymes. Twenty-two Gaucher disease mutations or created mutations were expressed in Spodoptera frugiperda (Sf9) cells and analyzed for catalytic properties, stability, inhibitor binding, and modifier interactions. Many Gaucher disease mutations encoded highly disruptive amino acid substitutions (e.g. P289L and D409V) and produced severely compromised proteins with very reduced activity (kcat < 1% of normal) and/or stability. Six mutant enzymes had sufficient catalytic activity (kcat approximately 5-30% of normal) for extensive studies. The highly conservative substitutions, i.e. F216Y or S364T and V394L, led to severe, but selective, abnormalities of enzyme stability or large decreases in catalytic activity, respectively. The T323I, N370S, and V394L enzymes interacted abnormally with active site-directed inhibitors and localized these residues to the glycon binding region. Selected mutant enzymes were poorly activated by phosphatidylserine (V394L, L444P, and R463C) or by saposin C (L444P and T323I), indicating that the enzyme sites for interaction with these activators were within the carboxyl one-third of the enzyme. Substitutions of Ser, Glu, and/or Gly at residues Asp-443 and/or Asp-445 demonstrated important steric roles for these residues in the active site, but neither is the catalytic nucleophile. Together with previous studies, the present analyses provide an insight into the pathogenesis of Gaucher disease and the functional organization of acid beta-glucosidase.

Amino Acid Sequence↗