Digestion of Starches by Soluble α-Amylase

Amylase has the distinction of being the first enzyme to be discovered. In 1833, Anselme Payen and Jean-François Persoz, chemists at a French sugar factory, extracted a substance from malt that converted starch to sugars. Payen and Persoz called this substance diastase (meaning a separation), because the enzyme converts the starch in barley seed to soluble sugars when the beer mash is heated, and this causes the husk to separate from the rest of the seed. The name diastase was subsequently changed to amylase, based on the Latin name for starch (amylum) and retaining the -ase suffix (now commonly used for naming enzymes) from the enzyme’s original name. Discovery of salivary and pancreatic amylases followed thereafter.

Production and secretion of amylase is restricted to the salivary and pancreatic exocrine glands. The amylases are α(1,4) endoglucosidases that cleave internal α(1,4) glycosidic bonds in linear starch chains to produce a variety of glucose oligomers. The salivary and pancreatic α-amylases are secreted as soluble proteins into the mouth or small intestine via the salivary ducts and pancreatic/common bile duct, respectively.

Thus digestion of starch begins in the mouth by action of salivary α-amylase as food is chewed and mixed with saliva. The action of amylase, however, ceases abruptly after the chewed food mixes with the acidic secretions of the stomach, because amylase activity requires neutral pH. Although the amylase protein is acid labile, the presence of starch in association with the amylase and the protection of amylase within the bolus of food provides some delay in amylase inactivation by the gastric acid (Rosenblum et al., 1988).

Pancreatic amylase is secreted in large quantities into the duodenal lumen. Most of the starch component of grains or legumes establishes contact with the pancreatic α-amylases within the polar bulk phase of the intestinal luminal milieu. The concentration of amylase achieved within the duodenal lumen greatly exceeds that required for cleavage of the bonds joining the glucose components of the starches. Indeed, the cleavage of starches to the final oligosaccharide products normally occurs in the uppermost part of the small intestine and is virtually complete by the time the meal reaches the duodenal–jejunal junction (Fogel and Gray, 1973). Furthermore, refined starches are hydrolyzed efficiently to glucose oligomers, even in patients with exocrine pancreatic insufficiency who have amylase levels that are only about 10% of normal (Fogel and Gray, 1973).

Differences in Structure and Digestion of Amylose and Amylopectin

It is important to consider the structure of the principal dietary starches, because amylase has specificity for only the α(1,4)-linked straight-chain regions of the glucosyl polysaccharide, whereas the most abundant food starches also have α(1,6)-branching links. The simplest starch is the linear and unbranched amylose, which is a polymer of α(1,4)-linked glucosyl units (∼600 glucose residues per molecule). Amylase has maximal specificity for the interior links, and its active site binds five consecutive glucose residues at specific subsites and cleaves between the second and third subsites, as shown in Figure 8-1, to form two smaller polymers. Sequential cleavage eventually leads to the production of pentasaccharides that bind with high affinity at all five of the amylase subsites. The pentasaccharide will be hydrolyzed at the penultimate linkage from the reducing end of the pentasaccharide to release the trisaccharide maltotriose and the disaccharide maltose. Products smaller than the pentasaccharide are not able to bind at all subsites and thus have very low affinity for the amylase active site; hence these smaller oligosaccharides are not readily cleaved by amylase. Overall, the sequential actions of amylase promote the release of maltotriose and maltose as the main final products of luminal amylose digestion.

Amylopectin is a more complex form of starch, representing about 80% of dietary polysaccharide. Figure 8-2 depicts the action of amylase on amylopectin to yield the final oligosaccharide products within the distal duodenal lumen. Although its linear segments are similar to those for amylose, amylopectin is branched by virtue of α(1,6) links positioned approximately every 25 residues along the chain. Glycogen, though a minor dietary carbohydrate, has a structure analogous to that of amylopectin and is also digested by amylase. Amylase is unable to cleave the α(1,6) branching link, and the structural angulation created by this linkage inhibits the enzyme from attacking some of the adjacent α(1,4) links. As a consequence, branched oligosaccharides called α-limit dextrins (or α-dextrins), along with maltose and maltotriose, are the final products of amylopectin hydrolysis by α-amylase. Dextrins of average mass of five to six glucose units represent nearly one third the mass of the final breakdown products of amylopectin.

Overall, the hydrolytic activities of salivary and pancreatic amylases on amylose and amylopectin result in production of glucose oligosaccharides and disaccharides containing α(1,4) linkages and glucose oligosaccharides containing both α(1,4) and α(1,6) linkages, with only about 4% of the glucose residues being released as free glucose monomers (Yook and Robyt, 2002). The subsequent steps of starch digestion are ensured by activities of the α-glucosidases associated with the apical membrane of the intestinal mucosal cells, as discussed later.

Regulation of Amylase Secretion

Both salivary and pancreatic amylase levels are low at birth but typically reach adult levels before 1 year of age. Expression of pancreatic amylase activity appears to lag behind the expression of other enzymes synthesized in the acinar cells of the pancreas. This has prompted some pediatricians and nutritionists to recommend withholding starch from the diet until about 6 months of age. Nevertheless, clinical symptoms resulting from cereal feeding before 6 months of age are rare (Lebenthal et al., 1983).

Secretion rate patterns for total protein and salivary amylase mirror those of salivary flow rate, suggesting there is little regulation of amylase synthesis or secretion by the salivary gland. Salivary secretion is mainly regulated by neural signals and responds to the sight, smell, and taste of food, which can increase salivary flow by up to eightfold.

In pancreatic acinar cells, amylase along with other digestive enzymes is packaged in large (<1 μm diameter) secretory vesicles known as zymogen granules that are stored in the acinar cells. In the acidic zymogen granule, amylase is inactive. In response to various secretagogues, such as cholecystokinin, or to neural signals, the contents of the zymogen granule undergo exocytosis into the lumen of the acinus, which feeds into the pancreatic duct. Upon release from the acidic zymogen granule into the pancreatic duct, amylase becomes active due to the higher pH of the pancreatic secretions. The higher pH of the pancreatic juice is largely due to the bicarbonate secreted by the pancreas. This bicarbonate secretion is important for neutralizing the acidic contents of the stomach as they enter the duodenum.

Completion of Starch Digestion by Integral Membrane Oligosaccharidases

The apical (brush border) membrane surface of the small intestinal enterocytes is replete with oligosaccharidases. Two of these oligosaccharidases are α-glucosidases that carry out the final steps in starch digestion (Semenza and Auricchio, 1991). Each one of these α-glucosidases, as synthesized, possesses two domains, with each domain containing an independent active site. The enzyme molecules are named for the main activities of the two domains: maltase–glucoamylase and sucrase–isomaltase. Both are attached to the apical membrane by an N-terminal hydrophobic sequence that spans the membrane.

Roles of Maltase–Glucoamylase and Sucrase–Isomaltase in Starch Digestion

These α-glucosidase activities are all classified as exoglucosidases because they can cleave one glucose residue at a time from the nonreducing end of starch oligosaccharides. All of these α-glucosidases cleave α(1,4) linkages of glucose residues. The functional differences among maltase–glucoamylase, sucrase, and isomaltase are the degree of their specificity for a particular substrate or glycosidic linkage at the nonreducing terminus of the glucose oligosaccharides. Glucoamylase has high specificity for the α(1,4) link at the nonreducing terminus of straight-chain glucosyl oligosaccharides containing from two to nine glucose units. Maltase hydrolyzes α(1,4) bonds in both starch oligosaccharides and in the disaccharide maltose. Sucrase displays high efficiency for the α(1,4) links of the smallest glucosyl oligosaccharides, maltose and maltotriose, and hence is an efficient maltase. It reinforces maltase–glucoamylase in the release of α(1,4)-linked glucose units, but its name comes from its unique capacity to cleave the link between the glucose and fructose units of sucrose [α-D-glucopyranosyl(1,2′)β′-D-fructofuranoside]. Similarly, isomaltase (α-dextrinase) has appreciable specificity for the α(1,4) links in the oligosaccharide products of starch digestion, but its maximal and unique specificity is for the α(1,6) branching link of the α-dextrins. Because of its α(1,6) specificity and the use of the α(1,6) disaccharide isomaltose as a substrate in the in vitro assay of its activity, it is commonly called isomaltase. Isomaltose, however, is not a natural substrate produced by α-amylase action on amylopectin. The term α-dextrinase is also used for isomaltase to more appropriately describe the enzyme’s capacity to cleave both α(1,4) and α(1,6) links in the oligosaccharide products (limit dextrins) of starch digestion.

Maltase–glucoamylase, isomaltase (α-dextrinase), and sucrase work in a complementary manner to cleave the bonds in the α-dextrins in sequence from the nonreducing end to release free glucose units. As shown in Figure 8-3, at each step in the process, one or more of these enzymes has high specificity for the α-glucosyl link closest to the nonreducing end of specific oligosaccharide products. These glucosidases produce free glucose for transport into the enterocyte.

Contributions of Maltase–Glucoamylase and Sucrase–Isomaltase to Oligosaccharide Hydrolysis

The relative contribution of maltase–glucoamylase versus sucrase–isomaltase in the hydrolysis of the α(1,4) linkages in the oligosaccharides released by action of amylase on dietary starch has historically been difficult to assess. The enzymatic activities of the two proteins overlap, and there is a lack of assays to completely distinguish the contributions of the two intestinal mucosal enzymes to starch digestion in humans as well as the inhibitory effect of starch digestion products on some of the catalytic domains.

Maltase–glucoamylase has a higher affinity (lower K_m) and faster rate of substrate turnover (higher k_cat) for hydrolysis of glucose oligomers than does sucrase–isomaltase, and thus it will catalyze a more rapid rate of hydrolysis at low concentrations of glucose oligomers (Table 8-2). However, it appears that maltase–glucoamylase contributes a relatively small fraction of the total glucose oligomer-hydrolyzing activity in humans in vivo. Indeed, sucrase–isomaltase activity in humans is about 20 times that of maltase–glucoamylase activity. Moreover, sucrase–isomaltase is not inhibited by lumenal starch-derived oligosaccharides as is the activity of maltase–glucoamylase in assays performed in vitro (Quezada-Calvillo et al., 2007). Thus sucrase–isomaltase probably contributes most of the α-glucosidase activity when high-starch diets are consumed.

Sucrose Digestion by Sucrase

Sucrose[α-D-glucopyranosyl(1,2′)β′-D-fructofuranoside], which is also known as table sugar, is the most abundantly consumed natural sweetener. Table sugar is refined from sugar cane or sugar beets, and various refined forms of sucrose are used in production of processed foods. Sucrose is also present in relatively large amounts in fruits, such as oranges and apples.

Intestinal digestion of sucrose requires hydrolysis of the α(1,2′)β′ linkage, which involves the anomeric carbon of both hexoses, to yield glucose and fructose. In mammalian species, the hydrolysis of sucrose is performed by the sucrase activity of the sucrase–isomaltase complex. The resulting monosaccharides are then absorbed by the intestinal epithelial cells.

Lactose Digestion by Lactase

Lactose [β-D-galactopyranosyl(1,4′)D-glucopyranoside] is a disaccharide abundant in the mammalian milk from which it takes its name (from the Latin lac, for “milk”). Milk ingestion by breast- or formula-fed infants provides approximately 70 g/L (or 55 g/day) of carbohydrates, of which approximately 60 g/L (or 47 g/day) is lactose. (The remainder of the milk carbohydrates are oligosaccharides that are not digestible by the human gastrointestinal enzymes.) The nutritional relevance of lactose is more limited after infancy because of the introduction of alternative dietary carbohydrates.

Lactose digestion in mammals requires lactase–phlorizin hydrolase activity, one of the intestinal disaccharidases present in the apical membrane of the small intestinal enterocytes. Although lactase–phlorizin hydrolase shows structural analogies with other α-glycosidases of the brush border membrane, it has a distinctive neutral β-galactosidic activity. The molecule contains two independent active sites, one responsible for lactase activity and the other associated with the phlorizin hydrolase activity. The β-galactosidase domain has high specificity for lactose. The physiological significance of the phlorizin hydrolase domain is unknown. It is named for its ability to hydrolyze phlorizin, which is a toxic 2′-glucoside of phloretin that occurs naturally in the bark and seeds of some fruit trees

Trehalose Digestion by Trehalase

Trehalose [α-D-glucopyranosyl-(1,1′)α′-D-glucopyranoside] is a disaccharide present in small amounts in bacteria, fungi, plants, and invertebrates (Elbein et al., 2003; Richards et al., 2002). Significant food sources of trehalose in modern Western diets include baker’s yeast, brewer’s yeast, cultivated and wild mushrooms, honey, sunflower seeds, sea algae, lobster, shrimp, and processed foods to which trehalose has been added. Trehalose may have constituted a larger source of sugar in the diet of ancient man, and some populations eat a much higher proportion of invertebrates (e.g., insects in which trehalose is the major hemolymph sugar) and fungi (e.g., yeasts, molds, mushrooms, and truffles) as part of their diets and therefore ingest more trehalose than is provided by typical Western diets. Reported concentrations of trehalose from natural sources vary because of experimental conditions, analytical methods, and the life stage of the organism assayed. In organisms, trehalose performs diverse functions that include energy storage and transport as well as protection against extreme temperatures or desiccation (Schiraldi et al., 2002).

The α(1,1′)α′ glucosidic linkage of the glucose residues in trehalose is hydrolyzed by trehalase, which is present in the brush border membrane of the intestinal epithelial cells of humans and most mammals. Hydrolysis of each molecule of trehalose by trehalase renders two molecules of free glucose. The presence of trehalase activity in the human intestine may reflect a widespread dependence of ancient humans on insects and fungi as primary sources of nutrients.

Effect of Developmental Stage and Diet on Saccharidase Expression

In humans the oligosaccharidases and disaccharidases are present in the gut before birth and are expressed throughout life. In general, levels of these enzymes are reduced in subjects who have fasted or are receiving parenteral nutrition, and levels are increased in subjects fed a carbohydrate-rich diet or in patients with uncontrolled diabetes. In most other mammals the lactase–phlorizin hydrolase is expressed from birth to weaning, and the other oligosaccharidases appear during the transition from maternal milk to starch-based diets. Lactase–phlorizin hydrolase in the intestinal mucosa of humans reaches maximal activity at an early postnatal age and then declines before adulthood in most people (Simon-Assmann et al., 1986). NF-LPH1 is a transcription factor that is present exclusively in intestinal epithelium and is thought to regulate expression of the lactase–phlorizin hydrolase gene. NF-LPH1 declines simultaneously with lactase–phlorizin hydrolase activity during the postweaning period. Thus a reduction in the expression of NF-LPH1 might be the cause of the decline in lactase activity that occurs before adulthood in most humans (Kuokkanen et al., 2003; Troelsen et al., 2003).

Crypt–Villus Expression of Saccharidases

Expression of oligosaccharidase and disaccharidase activities at the cellular level is tightly linked to enterocyte differentiation (Boudreau et al., 2002; Troelsen et al., 1997). For example, immature enterocytes developing from stem cells in crypts of the intestinal mucosa do not have sucrase–isomaltase activity. As enterocytes traverse to the crypt–villus junction, sucrase–isomaltase messenger RNA (mRNA) and sucrose–isomaltase activity are observed to increase. The level of sucrase–isomaltase mRNA reaches its peak when enterocytes are located in the lower and mid villus region and then progressively decreases as these cells move toward the tip (Traber et al., 1992). Expression of other disaccharidase and oligosaccharidase activities follows a similar pattern.

Posttranslational Processing of Saccharidases

The posttranslational processing of newly synthesized oligosaccharidases and disaccharidases has been the subject of a number of studies. The general pathways, summarized in Figure 8-5, are similar for lactase–phlorizin hydrolase, sucrase–isomaltase, and maltase–glucoamylase, although each has some specific and unique characteristics (Weisz and Rodriguez-Boulan, 2009). Trehalase expression and processing has little similarity to that of the other disaccharidases. All of these oligosaccharidases and disaccharidases are synthesized in the enterocyte where they are processed in the endoplasmic reticulum (ER) and Golgi apparatus with final vectorial transport to the apical membrane. Lactase–phlorizin hydrolase, sucrase–isomaltase, and maltase–glucoamylase all undergo extensive N– and O-glycosylation in the ER and Golgi apparatus, but trehalase has only N-glycosylation sites. The orientation of these enzymes in the membrane is such that the oligosaccharidase domains, including the active catalytic sites, are on the luminal side of the apical membrane and thus available for efficient cleavage of the luminal substrates.

Maltase–isomaltase and sucrase–isomaltase are both anchored in the membrane by an N-terminal hydrophobic sequence, whereas lactase–phlorizin hydrolase is inserted into the plasma membrane by its C-terminus, as illustrated in Figure 8-4. In contrast to the other disaccharidases, the C-terminus of trehalase is anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Proteolysis is involved in production of the final mature forms of sucrase–isomaltase and lactase–phlorizin hydrolase, whereas no proteolytic processing has been documented to occur for human maltase–glucoamylase.

The human lactase–phlorizin hydrolase gene encodes a polypeptide of four domains, but two of these domains are eliminated during the maturation process to render a mature protein that has two domains containing one potential active site each. Proteolytic processing occurs intracellularly, and proteolytic trimming by pancreatic proteases in the small intestine occurs at a second site after the enzyme has been transferred to the plasma membrane. The N-terminal domains of lactase–phlorizin hydrolase, which are intracellularly cleaved to yield the active brush border enzyme, are required as a chaperone for exit of the mature polypeptide from the ER. The phlorizin hydrolase domain appears to serve an additional chaperone role and is also required for correct folding and trafficking of the lactase domain out of the ER (Behrendt et al., 2010). The O-glycosylation that is added in the Golgi apparatus is required for sorting of lactase−phlorizin hydrolase to the luminal membrane.

Sucrase–isomaltase is transported via the vesicular system to the brush border membrane of enterocytes where it is then subjected to extracellular processing by pancreatic proteolytic enzymes present in the intestinal lumen. The molecule is cleaved to generate a membrane-bound N-terminal isomaltase subunit and a free C-terminal sucrase subunit, which remains associated with the N-terminal isomaltase domain through noncovalent interactions. The C-terminal domain of sucrase–isomaltase appears to act as an intramolecular chaperone for the isomaltase domain and is required for the protein to exit the ER and for subsequent proteolytic processing of the enzyme. In the absence of the sucrase domain, isomaltase remains associated with the ER through its association with the folding chaperone calnexin and is not processed properly (Jacob et al., 2002).