Β-galactosidase called lactase, beta-gal or β-gal, is a glycoside hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides through the breaking of a glycosidic bond. Β-galactosides include carbohydrates containing galactose where the glycosidic bond lies above the galactose molecule. Substrates of different β-galactosidases include ganglioside GM1, lactosylceramides and various glycoproteins. Β-galactosidase is an exoglycosidase which hydrolyzes the β-glycosidic bond formed between a galactose and its organic moiety. It may cleave fucosides and arabinosides but with much lower efficiency, it is an essential enzyme in the human body. Deficiencies in the protein can result in Morquio B syndrome. In E. coli, the lacZ gene is the structural gene for β-galactosidase. Β-galactosidase synthesis stops. Beta-galactosidase has many homologues based on similar sequences. A few are evolved beta-galactosidase, beta-glucosidase, 6-phospho-beta-galactosidase, beta-mannosidase, lactase-phlorizin hydrolase.
Although they may be structurally similar, they all have different functions. Beta-gal is inhibited by L-ribose, non-competitive inhibitor iodine, competitive inhibitors phenylthyl thio-beta-D-galactoside, D-galactonolactone, isopropyl thio-beta-D-galactoside, galactose.β-galactosidase is important for organisms as it is a key provider in the production of energy and a source of carbons through the break down of lactose to galactose and glucose. It is important for the lactose intolerant community as it is responsible for making lactose-free milk and other dairy products. Many adult humans lack the lactase enzyme, which has the same function of beta-gal, so they are not able to properly digest dairy products. Beta-galactose is used in such dairy products as yogurt, sour cream, some cheeses which are treated with the enzyme to break down any lactose before human consumption. In recent years, beta-galactosidase has been researched as a potential treatment for lactose intolerance through gene replacement therapy where it could be placed into the human DNA so individuals can break down lactose on their own.
The 1,024 amino acids of E. coli β-galactosidase were first sequenced in 1970, its structure determined twenty-four years in 1994. The protein is a 464-kDa homotetramer with 2,2,2-point symmetry; each unit of β-galactosidase consists of five domains. The third domain contains the active site; the active site is made up of elements from two subunits of the tetramer, disassociation of the tetramer into dimers removes critical elements of the active site. The amino-terminal sequence of β-galactosidase, the α-peptide involved in α-complementation, participates in a subunit interface, its residues 22-31 help to stabilize a four-helix bundle which forms the major part of that interface, residue 13 and 15 contributing to the activating interface. These structural features provide a rationale for the phenomenon of α-complementation, where the deletion of the amino-terminal segment results in the formation of an inactive dimer. Β-galactosidase can catalyze two different reactions in organisms. In one, it can go through a process called transgalactosylation to make allolactose, creating a positive feedback loop for the production of β-gal.
It can hydrolyze lactose into galactose and glucose which will proceed into glycolysis. The active site of β-galactosidase catalyzes the hydrolysis of its disaccharide substrate via "shallow" and "deep" binding. Galactosides such as PETG and IPTG will bind in the shallow site when the enzyme is in "open" conformation while transition state analogs such as L-ribose and D-galactonolactone will bind in the deep site when the conformation is "closed"; the enzymatic reaction consists of two chemical steps and degalactosylation. Galactosylation is the first chemical step in the reaction where Glu461 donates a proton to a glycosidic oxygen, resulting in galactose covalently bonding with Glu537. In the second step, degalactosylation, the covalent bond is broken when Glu461 accepts a proton, replacing the galactose with water. Two transition states occur in the deep site of the enzyme during the reaction, once after each step; when water participates in the reaction, galactose is formed, when D-glucose acts as the acceptor in the second step, transgalactosylation occurs.
It has been kinetically measured that single tetramers of the protein catalyze reactions at a rate of 38,500 ± 900 reactions per minute. Monovalent potassium ions as well as divalent magnesium ions are required for the enzyme's optimal activity; the beta-linkage of the substrate is cleaved by a terminal carboxyl group on the side chain of a glutamic acid. In E. coli, Glu-461 was thought to be the nucleophile in the substitution reaction. However, it is now known. Instead, Glu-537 is the actual nucleophile, binding to a galactosyl intermediate. In humans, the nucleophile of the hydrolysis reaction is Glu-268. Gly794 is important for β-gal activity, it is responsible for putting the enzyme in a "closed", ligand bounded, conformation or "open" conformation, acting like a "hinge" for the active site loop. The different conformations ensure. In the presence of a slow substrate, Gly794 activity increased as well as an increase in galactosylation and decrease in degalactosylation
Hexosaminidase is an enzyme involved in the hydrolysis of terminal N-acetyl-D-hexosamine residues in N-acetyl-β-D-hexosaminides. Functional lysosomal β-hexosaminidase enzymes are dimeric in structure. Three isozymes are produced through the combination of α and β subunits to form any one of three active dimers: The α and β subunits are encoded by separate genes, HEXA and HEXB respectively. Beta-hexosaminidase and the cofactor GM2 activator protein catalyze the degradation of the GM2 gangliosides and other molecules containing terminal N-acetyl hexosamines. Gene mutations in HEXB result in Sandhoff disease. Though the alpha and beta subunits of lysosomal hexosaminidase can both cleave GalNAc residues, only the alpha subunit is able to hydrolyze GM2 gangliosides because of a key residue, Arg-424, a loop structure that forms from the amino acid sequence in the alpha subunit; the loop in the alpha subunit, consisting of Gly-280, Ser-281, Glu-282, Pro-283, absent in the beta subunit, serves as an ideal structure for the binding of the GM2 activator protein, arginine is essential for binding the N-acetyl-neuraminic acid residue of GM2 gangliosides.
The GM2 activator protein transports GM2 gangliosides and presents the lipids to hexosaminidase, so a functional hexosaminidase enzyme is able to hydrolyze GM2 gangliosides into GM3 gangliosides by removing the N-acetylgalactosamine residue from GM2 gangliosides. A Michaelis complex consisting of a glutamate residue, a GalNAc residue on the GM2 ganglioside, an aspartate residue leads to the formation of an oxazolinium ion intermediate. A glutamate residue works as an acid by donating its hydrogen to the glycosidic oxygen atom on the GalNAc residue. An aspartate residue positions the C2-acetamindo group so that it can be attacked by the nucleophile; the aspartate residue stabilizes the positive charge on the nitrogen atom in the oxazolinium ion intermediate. Following the formation of the oxazolinium ion intermediate, water attacks the electrophillic acetal carbon. Glutamate acts as a base by deprotonating the water leading to the formation of the product complex and the GM3ganglioside. There are numerous mutations that lead to hexosaminidase deficiency including gene deletions, nonsense mutations, missense mutations.
Tay–Sachs disease occurs when hexosaminidase A loses its ability to function. People with Tay–Sachs disease are unable to remove the GalNAc residue from the GM2 ganglioside, as a result, they end up storing 100 to 1000 times more GM2 gangliosides in the brain than the normal person. Over 100 different mutations have been discovered just in infantile cases of Tay–Sachs disease alone; the most common mutation, which occurs in over 80 percent of Tay–Sachs patients, results from a four base pair addition in exon 11 of the Hex A gene. This insertion leads to an early stop codon. Children born with Tay–Sachs die between two and four years of age from aspiration and pneumonia. Tay–Sachs causes cerebral degeneration and blindness. Patients experience flaccid extremities and seizures. At this point in time, there has been no effective treatment of Tay -- Sachs disease. NAG-thiazoline, NGT, acts as mechanism based inhibitor of hexosaminidase A. In patients with Tay–Sachs disease, NGT acts as a molecular chaperone by binding in the active site of hexosaminidase A which helps create a properly folded hexosaminidase A.
The stable dimer conformation of hexosaminidase A has the ability to leave the endoplasmic reticulum and is directed to the lysosome where it can perform the degradation of GM2 gangliosides. The two subunits of hexosaminidase A are shown below: The bifunctional protein NCOAT, encoded by the MGEA5 gene possesses both hexosaminidase and histone acetyltransferase activities. NCOAT is known as hexosaminidase C and has distinct substrate specificities compared to lysosomal hexosaminidase A. A single-nucleotide polymorphism in the human O-GlcNAcase gene is linked to diabetes mellitus type 2. A fourth mammalian hexosaminidase polypeptide, designated hexosaminidase D has been identified. GeneReviews/NCBI/NIH/UW entry on hexosaminidase A deficiency, Tay–Sachs disease hexosaminidase A at the US National Library of Medicine Medical Subject Headings EC 126.96.36.199
Manihot esculenta called cassava, yuca, mandioca and Brazilian arrowroot, is a woody shrub native to South America of the spurge family, Euphorbiaceae. It is extensively cultivated as an annual crop in tropical and subtropical regions for its edible starchy tuberous root, a major source of carbohydrates. Though it is called yuca in Spanish and in the United States, it is not related to yucca, a shrub in the family Asparagaceae. Cassava, when dried to a powdery extract, is called tapioca. Cassava is the third-largest source of food carbohydrates in the tropics, after maize. Cassava is a major staple food in the developing world, providing a basic diet for over half a billion people, it is one of the most drought-tolerant crops, capable of growing on marginal soils. Nigeria is the world's largest producer of cassava, while Thailand is the largest exporter of dried cassava. Cassava is classified as either bitter. Like other roots and tubers, both bitter and sweet varieties of cassava contain antinutritional factors and toxins, with the bitter varieties containing much larger amounts.
It must be properly prepared before consumption, as improper preparation of cassava can leave enough residual cyanide to cause acute cyanide intoxication and ataxia, partial paralysis, or death. The more toxic varieties of cassava are a fall-back resource in times of famine or food insecurity in some places. Farmers prefer the bitter varieties because they deter pests and thieves; the cassava root is long and tapered, with a firm, homogeneous flesh encased in a detachable rind, about 1 mm thick and brown on the outside. Commercial cultivars can be 5 to 10 cm in diameter at the top, around 15 to 30 cm long. A woody vascular bundle runs along the root's axis; the flesh can be yellowish. Cassava roots are rich in starch and contain small amounts of calcium and vitamin C. However, they are poor in protein and other nutrients. In contrast, cassava leaves are a good source of protein, but deficient in the amino acid methionine and tryptophan. Wild populations of M. esculenta subspecies flabellifolia, shown to be the progenitor of domesticated cassava, are centered in west-central Brazil, where it was first domesticated no more than 10,000 years BP.
Forms of the modern domesticated species can be found growing in the wild in the south of Brazil. By 4,600 BC, manioc pollen appears in the Gulf of Mexico lowlands, at the San Andrés archaeological site; the oldest direct evidence of cassava cultivation comes from a 1,400-year-old Maya site, Joya de Cerén, in El Salvador. With its high food potential, it had become a staple food of the native populations of northern South America, southern Mesoamerica, the Caribbean by the time of European contact in 1492. Cassava was a staple food of pre-Columbian peoples in the Americas and is portrayed in indigenous art; the Moche people depicted yuca in their ceramics. Spaniards in their early occupation of Caribbean islands did not want to eat cassava or maize, which they considered insubstantial and not nutritious, they much preferred foods from Spain wheat bread, olive oil, red wine, meat, considered maize and cassava damaging to Europeans. For these Christians in the New World, cassava was not suitable for communion since it could not undergo transubstantiation and become the body of Christ.
"Wheat flour was the symbol of Christianity itself" and colonial-era catechisms stated explicitly that only wheat flour could be used. The cultivation and consumption of cassava was nonetheless continued in both Portuguese and Spanish America. Mass production of cassava bread became the first Cuban industry established by the Spanish, Ships departing to Europe from Cuban ports such as Havana, Santiago and Baracoa carried goods to Spain, but sailors needed to be provisioned for the voyage; the Spanish needed to replenish their boats with dried meat, water and large amounts of cassava bread. Sailors complained. Tropical Cuban weather was not suitable for wheat planting and cassava would not go stale as as regular bread. Cassava was introduced to Africa by Portuguese traders from Brazil in the 16th century. Around the same period, it was introduced to Asia through Columbian Exchange by Portuguese and Spanish traders, planted in their colonies in Goa, Eastern Indonesia and the Philippines. Maize and cassava are now important staple foods.
Cassava has become an important staple in Asia, extensively cultivated in Indonesia and Vietnam. Cassava is sometimes described as the "bread of the tropics" but should not be confused with the tropical and equatorial bread tree, the breadfruit or the African breadfruit. In 2016, global production of cassava root was 277 million tonnes, with Nigeria as the world's largest producer having 21% of the world total. Other major growers were Thailand and Indonesia. Cassava is one of the most drought-tolerant crops, can be grown on marginal soils, gives reasonable yields where many other crops do not grow well. Cassava is well adapted within latitudes 30° north and south of the equator, at elevations between sea level and 2,000 m above sea level, in equatorial temperatures, with rainfalls from 50 mm to 5 m annually, to poor soils with a pH ranging from acidic to alkaline; these conditions are common in certain parts of Africa and So
Alpha-glucosidase is a glucosidase located in the brush border of the small intestine that acts upon α bonds. This is in contrast to beta-glucosidase. Alpha-glucosidase breaks down starch and disaccharides to glucose. Maltase, a similar enzyme that cleaves maltose, is nearly functionally equivalent. Other glucosidases include: Cellulase Beta-glucosidase Debranching enzyme Alpha-glucosidase hydrolyzes terminal non-reducing -linked alpha-glucose residues to release a single alpha-glucose molecule. Alpha-glucosidase is a carbohydrate-hydrolase that releases alpha-glucose as opposed to beta-glucose. Beta-glucose residues can be released by a functionally similar enzyme; the substrate selectivity of alpha-glucosidase is due to subsite affinities of the enzyme’s active site. Two proposed mechanisms include an oxocarbenium ion intermediate. Rhodnius prolixus, a blood-sucking insect, forms hemozoin during digestion of host hemoglobin. Hemozoin synthesis is dependent on the substrate binding site of alpha-glucosidase.
Trout liver alpha-glucosidases were characterized. It was shown that for one of the trout liver alpha-glucosidases maximum activity of the enzyme was increased by 80% during exercise in comparison to a resting trout; this change was shown to correlate to an activity increase for liver glycogen phosphorylase. It is proposed that alpha-glucosidase in the glucosidic path plays an important part in complementing the phosphorolytic pathway in the liver’s metabolic response to energy demands of exercise. Yeast and rat small intestinal alpha-glucosidases have been shown to be inhibited by several groups of flavonoids. Alpha-glucosidases can be divided, into two families; the gene coding for human lysosomal alpha-glucosidase is about 20 kb long and its structure has been cloned and confirmed. Human lysosomal alpha-glucosidase has been studied for the significance of the Asp-518 and other residues in proximity of the enzyme’s active site, it was found that substituting Asp-513 with Glu-513 interferes with posttranslational modification and intracellular transport of alpha-glucosidase’s precursor.
Additionally, the Trp-516 and Asp-518 residues have been deemed critical for the enzyme’s catalytic functionality. Kinetic changes in alpha-glucosidase have been shown to be induced by denaturants such as guanidinium chloride and SDS solutions; these denaturants cause loss of conformational change. A loss of enzyme activity occurs at much lower concentrations of denaturant than required for conformational changes; this leads to a conclusion that the enzyme’s active site conformation is less stable than the whole enzyme conformation in response to the two denaturants. Glycogen storage disease type II called Pompe disease: a disorder in which alpha-glucosidase is deficient. In 2006, the drug alglucosidase alfa became the first released treatment for Pompe disease and acts as an analog to alpha-glucosidase. Further studies of alglucosidase alfa revealed that iminosugars exhibit inhibition of the enzyme, it was found. It was shown that 1-deoxynojirimycin would bind the strongest of the sugars tested and blocked the active site of the enzyme entirely.
The studies enhanced knowledge of the mechanism. Diabetes: Acarbose, an alpha-glucosidase inhibitor and reversibly inhibits alpha-glucosidase in the intestines; this inhibition lowers the rate of glucose absorption through delayed carbohydrate digestion and extended digestion time. Acarbose may be able to prevent the development of diabetic symptoms. Hence, alpha-glucosidase inhibitors are used as anti-diabetic drugs in combination with other anti-diabetic drugs. Luteolin has been found to be a strong inhibitor of alpha-glucosidase; the compound can inhibit the enzyme up to 36% with a concentration of 0.5 mg/ml. As of 2016, this substance is being tested in rats and cell culture. Flavonoid analogues have been demonstrated with inhibition activity. Azoospermia: Diagnosis of azoospermia has potential to be aided by measurement of alpha-glucosidase activity in seminal plasma. Activity in the seminal plasma corresponds to the functionality of the epididymis. Antiviral agents: Many animal viruses possess an outer envelope composed of viral glycoproteins.
These are required for the viral life cycle and utilize cellular machinery for synthesis. Inhibitors of alpha-glucosidase show that the enzyme is involved in the pathway for N-glycans for viruses such as HIV and human hepatitis B virus. Inhibition of alpha-glucosidase can prevent fusion of HIV and secretion of HBV. Alglucosidase alfa Alpha-glucosidase inhibitor
Chitinases are hydrolytic enzymes that break down glycosidic bonds in chitin. As chitin is a component of the cell walls of fungi and exoskeletal elements of some animals, chitinases are found in organisms that either need to reshape their own chitin or dissolve and digest the chitin of fungi or animals. Chitinivorous organisms include many bacteria, which may be detritivorous, they attack living arthropods, zooplankton or fungi or they may degrade the remains of these organisms. Fungi, such as Coccidioides immitis possess degradative chitinases related to their role as detritivores and to their potential as arthropod pathogens. Chitinases are present in plants. Expression is mediated by the NPR1 gene and the salicylic acid pathway, both involved in resistance to fungal and insect attack. Other plant chitinases may be required for creating fungal symbioses. Although mammals do not produce chitin, they have two functional chitinases and acidic mammalian chitinase, as well as chitinase-like proteins that have high sequence similarity but lack chitinase activity.
Endochitinases randomly split chitin at internal sites of the chitin microfibril, forming soluble, low molecular mass multimer products. The multimer products includes di-acetylchitobiose and chitotetraose, with the dimer being the predominant product. Exochitinases have been divided into two sub categories: Chitobiosidases act on the non-reducing end of the chitin microfibril, releasing the dimer, di-acetylchitobiose, one by one from the chitin chain. Therefore, there is no release of oligosaccharides in this reaction. Β-1,4- N-acetylglucosaminidases split the multimer products, such as di-acetylchitobiose and chitotetraose, into monomers of N-acetylglucoseamine. Chitinases were classified based on the amino acid sequences, as that would be more helpful in understanding the evolutionary relationships of these enzymes to each other. Therefore, the chitinases were grouped into three families: 18, 19, 20. Both families 18 and 19 consists of endochitinases from a variety of different organisms, including viruses, fungi and plants.
However, family 19 comprises plant chitinases. Family 20 includes a similar enzyme, N-acetylhexosaminidase, and as the gene sequences of the chitinases were known, they were further classified into six classes based on their sequences. Characteristics that determined the classes of chitinases were the N-terminal sequence, localization of the enzyme, isoelectric pH, signal peptide, inducers. Class I chitinases had a cysteine-rich N-terminal, leucine- or valine-rich signal peptide, vacuolar localization, and Class I chitinases were further subdivided based on their acidic or basic nature into Class Ia and Class Ib, respectively. Class 1 chitinases were found to comprise only plant chitinases and endochitinases. Class II chitinases did not have the cysteine-rich N-terminal but had a similar sequence to Class I chitinases. Class II chitinases were found in plants and bacteria and consisted of exochitinases. Class III chitinases did not have similar sequences to chitinases in Class I or Class II. Class IV chitinases had similar characteristics, including the immunological properties, as Class I chitinases.
However, Class IV chitinases were smaller in size compared to Class I chitinases. Class V and Class VI chitinases are not well characterized. However, one example of a Class V chitinase showed two chitin binding domains in tandem, based on the gene sequence, the cysteine-rich N-terminal seemed to have been lost during evolution due to less selection pressure that caused the catalytic domain to lose its function. Like cellulose, chitin is an abundant biopolymer, resistant to degradation, it is not digested by animals, though certain fish are able to digest chitin. It is assumed that chitin digestion by animals requires bacterial symbionts and lengthy fermentations, similar to cellulase digestion by ruminants. Chitinases have been isolated from the stomachs of certain mammals, including humans. Chitinase activity can be detected in human blood and cartilage; as in plant chitinases this may be related to pathogen resistance. Chitinases produced in the human body may be related in response to allergies, asthma has been linked to enhanced chitinase expression levels.
Human chitinases may explain the link between some of the most common allergies and worm infections, as part of one version of the hygiene hypothesis. The link between chitinases and salicylic acid in plants is well established—but there is a hypothetical link between salicylic acid and allergies in humans. Regulation varies from species to species, within an organism, chitinases with different physiological functions would be under different regulation mechanisms. For example, chitinases that are involved in maintenance, such as remodeling the cell wall, are constitutively expressed. However, chitinases that have specialized function
Beta-glucosidase catalyzes the hydrolysis of the glycosidic bonds to terminal non-reducing residues in beta-D-glucosides and oligosaccharides, with release of glucose. Synonyms and related enzymes include gentiobiase, emulsin, aryl-beta-glucosidase, beta-D-glucosidase, beta-glucoside glucohydrolase, amygdalinase, p-nitrophenyl beta-glucosidase, amygdalase, linamarase and beta-1,6-glucosidase. Cellulose is a polymer composed of beta-1,4-linked glucosyl residues. Cellulases and beta-glucosidases are required by organisms that can consume it; these enzymes are powerful tools for degradation of plant cell walls by pathogens and other organisms consuming plant biomass. Amygdalin beta-glucosidase Cellulase, a suite of enzymes produced chiefly by fungi and protozoans that catalyze cellulolysis Glucosylceramidase, a related enzyme Prunasin beta-glucosidase Vicianin beta-glucosidase beta-Glucosidase at the US National Library of Medicine Medical Subject Headings GO-database listing'GO:0016162 cellulose 1,4-beta-cellobiosidase activity' Risk Assessment Summary, CEPA 1999.
Trichoderma reesei P59G