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Digestive enzymes [email protected] From food intake to absorption Mechanical homogenization of food, mixing with fluids secreted by the glands of GIT Secretion of digestive enzymes that hydrolyze macromolecules to oligomers/monomers Secretion of electrolytes, acid, or base to provide an appropriate environment for enzyme digestion Secretion of bile acids to solubilize lipids Hydrolysis of oligomers by intestinal surface enzymes Transport of nutrient molecules and of electrolytes from the intestinal lumen across the epithelial cells into blood or lymph Function of particular organs in digestion and absorption Pancreas is the major organ that synthesizes the digestive enzymes Small intestine is a principal site of digestion and absorption In the small intestine, there are three compartments where digestion and absorption occurs: 1) Pancreatic enzymes together with bile are poured into the lumen of the descending part of the duodenum bulk of the intra-luminal digestion occurs distal to this site 2) Digestion of oligomers of AA and saccharides is accomplished by the enzymes in the luminal plasma membranes of chief epithelial cells, enterocytes (i.e. in the microvilli forming the brush border); these enzymes – usually glycoproteins 3) Intracellular digestion in the cytoplasm of enterocytes is of some importance for hydrolysis of di- and tripeptides Zymogens Digestive enzymes are usually synthesized as larger inactive precursors – zymogens Otherwise they would digest the tissues that synthesize them: acute pancreatitis is characterized by the premature activation of the digestive enzymes produced by this gland → auto-digestion of the pancreas Synthesis of zymogens Proteins destined for secretion are synthesized on polysomes of the RER Their N-terminus contains a signal sequence that anchors ribosome to the membrane of ER → release of the protein into ER; then, the signal sequence may be clipped off The protein is transported to the Golgi complex; in ER and GC, glycosylation may occur The proteins are stored in vesicles After stimulus, granules move to the luminal plasma membrane (PM), where their membranes fuse with the PM, releasing the content into the lumen…exocytosis Zymogens are activated by proteolysis Proenzymes (zymogens) are activated by proteolytic cleavage in the lumen of the GIT: pepsinogen trypsinogen chymotrypsinogen proelastase procarboxypeptidases prophospholipases Activation of pepsinogen Pepsinogen is secreted from the cells of the stomach Pepsinogen is activated by the proteolytic removal of 44 AAs from its N-terminus – either as an intramolecular reaction (autoactivation) or by active pepsin (autocatalysis) This reaction takes place at pH values below 5 Activation of pancreatic zymogens in the lumen of the small intestine chymotrypsinogen, proelastase, procarboxypeptidases, prophospholipase enteropeptidase (produced in duodenum) trypsinogen trypsin – 6 N-terminal AAs autocatalytic activation by the same cleavage by active trypsin chymotrypsin, elastase, carboxypeptidases, phospholipase Trypsin activates the other pancreatic zymogens as well as itself!!! „Strategies“ that prevent premature zymogen activation At pH>2, the peptide (44 AA) clipped of pepsinogen remains bound to pepsin, masking its active site, i.e. acting like an inhibitor; it is released by a drop of pH below 2 or further degradation by pepsin Pancreatic trypsin inhibitor, a small polypeptide, blocks any trypsin that is erroneously activated within the pancreas Regulation of secretion Through secretagogues that interact with the receptors on the surface of the exocrine cells → signal cascade leading to fusion of granules with PM Organ Secretion Secretagogue Salivary gland NaCl, amylase Stomach HCl, pepsinogen acetylcholine, histamine, gastrin (peptide) Pancreas NaCl, enzymes acetylcholine, cholecystokinin (peptide secreted by cells of small int. after stimulation by AA and peptides from gastric proteolysis, by FAs, and by acid pH) NaHCO3, NaCl secretin (peptide secreted by cells of small int.; secretion stimulated by luminal pH < 5) acetylcholine DIGESTION OF PROTEINS By peptidases (proteases): endopeptidases – attack internal bonds: • pepsin • trypsin • chymotrypsin • elastase exopeptidases – cleave off 1 AA at a time from: • C-terminus – carboxypeptidases • N-terminus – aminopeptidases Revision – types of peptidases based on the groups in the active site Type Active site pH optimum Serine proteases Ser, His, Asp 7-9 Cysteine proteases Cys, His 3-6 Aspartate proteases 2 x Asp 2-5 Metalloproteases Zn2+ (coordinated to amino acids) 7-9 Peptidases = hydrolases with specificity for the peptide bond …exhibiting different substrate specificity: Pepsins Role: gastric digestion of proteins Acid in the stomach serves to kill off microorganisms and to denature proteins (denaturation makes proteins more susceptible to proteolysis) Pepsins are acid stable and pH optimum is about 2!!! Major products of pepsin action: larger peptide fragments and some free AAs; this mix = peptone Importance lies mainly in generation of peptides and AAs that stimulate cholecystokinin release in the duodenum Pancreatic enzymes trypsin chymotrypsin elastase carboxypeptidases Active at neutral pH depend on neutralization of gastric HCl by pancreatic NaHCO3 The combined action of pancreatic peptidases results in the formation of free AAs and small peptides (2-8 AA) Intestinal peptidases digest small peptides to AAs Luminal surface of intestinal epithelial cell contains endopeptidases, aminopeptidases, and dipeptidases that cleave oligopeptides released by pancreatic peptidases End products: free AA, di- and tripeptides → absorbed by the epithelial cells (enterocytes) via specific amino acid or peptide transport systems Di- and tripeptides are hydrolyzed by intestinal cytoplasmic peptidases AAs are transported into interstitial space and absorbed into the portal blood DIGESTION OF SACCHARIDES 1) Polysaccharides (starch, glycogen) are attacked by -amylase, which is present in saliva and pancreatic juice (more important) -amylase attacks the internal -1,4-glucosidic bonds (-1,6-bonds are not attacked) products: maltose, maltotriose, -limit dextrins (8 glucose units on average and one or more -1,6-bonds) 2) Final hydrolysis of oligosaccharides is carried out by surface enzymes of the small intestinal epithelial cells – disaccharidases and oligosaccharidases These enzymes – often exoglycosidases (clip-off one monosaccharide at a time from a non-reducing end) Saccharide absorption End products: monosaccharides, mainly D-glucose, D-galactose, D-fructose These are transported by a carrier-mediated process into enterocytes and then into the blood of the portal venous system Not everything can be digested Many plant polymers, including celulloses, hemicelluloses, inulin, pectin, are resistant to human digestive enzymes A small percentage of this „dietary fiber“ is hydrolyzed and then anaerobically metabolized by the bacteria of the lower intestinal tract (bacteria possess more types of saccharidases) This bacterial fermentation produces H2, CH4, CO2, H2S, acetate, propionate, butyrate, lactate Lactase deficiency Experienced as milk intolerance Cause: a) genetic defect b) decline of lactase activity with age c) decline of activity due to an intestinal disease Consequences: inability to absorb lactose accumulation of undigested lactose, bacterial fermentation of lactose production of gas (distension of gut, flatulence) and osmotically active solutes that draw water into the intestinal lumen (diarrhea) Lysozyme Hydrolyzes -1,4-glycosidic bonds in the bacterial cell wall polysaccharide peptidoglycan Kills only some types of bacteria DIGESTION OF LIPIDS Lipids – sparingly or not at all soluble in aqueous solutions Two problems have to be overcome: poor accessibility of the substrate to the enzyme aggregation of products of hydrolysis to larger complexes that are hard to absorb Phases of lipid digestion and absorption Hydrolysis of TGs to FFAs and monoacylglycerols Solubilization of the products and their transport from the intestinal lumen to the cell surface Uptake of FFAs and monoacylglycerols into the cell and resynthesis to TGs Packaging of TGs into chylomicrons Exocytosis of chylomicrons, release into lymph Digestion of lipids is initiated in stomach In stomach, acid-stable lipase, secreted by stomach (gastric lipase) and in trace amounts by lingual glands (lingual lipase), converts TGs mostly into FAs and 1,2diacylglycerols (small amount of monoacylglycerols is also produced) Importance: TGs are converted to products that possess both polar and non-polar groups, and therefore act as surfactants: adsorb to water-lipid interface and stabilize it dispersion of the lipid phase into smaller droplets (emulsification) better availability of the substrate to the lipases, including pancreatic lipase as well Among dietary lipids, FAs, monoacylglycerols, and phospholipids are the major surfactants Importance in infants: these lipases have the unique ability to initiate the degradation of maternal milk fat globules Major enzyme for TG hydrolysis is the pancreatic lipase Pancreatic lipase binds to the fat droplets and cleaves TGs, mainly to FFAs and 2-monoacylglycerols The enzyme requires „pretreatment“ of the substrate by preduodenal lipase and its solubilization (by FAs, phospholipids, monoacylglycerols) The enzyme also requires colipase (secreted by the pancreas) that binds both to the interface and to lipase, thereby anchoring and activating the enzyme Absorption of resulting FAs and monoacyl- glycerols requires bile salts micelles Digestion of phospholipids By phospholipases, especially by phospholipase A2 (requires bile acids for activity): FAs and lysophospholipids are absorbed from the bile acid micelles In the intestinal mucosa, the absorbed lysophospholipids are reacylated with acyl-CoA Hydrolysis of cholesterol esters By pancreatic cholesterol esterase The free cholesterol is transported in the bile acid micelles and absorbed through the brush border Here, it is reacylated with acyl-CoA Bile acid micelles solubilize lipids Primary bile acids are synthesized by the liver and in peroxisomes, they are conjugated with glycine or taurine (H2N-CH2CH2SO3-) In the bile, the bile acids and their conjugates are in a salt (anionic) form – that is why they are also called bile salts A portion of the primary bile acids in the intestine is subjected to the modifications by intestinal bacteria → secondary bile acids Bile salts are secreted with the bile into the duodenum. Primary and secondary bile acids are reabsorbed by the ileum into the portal blood, taken up by the liver, and then resecreted into the bile…enterohepatic circulation Bile acid has a hydrophobic surface and a hydrophilic surface The most abundant bile salt in humans – glycocholate: Bile acid micelles Hydrophobic region of bile salt is oriented FROM the water molecules whereas hydrophilic region interacts with water Mixed micelles contain (beside bile acids) phospholipids, cholesterol, FAs, and acylglycerols, that should be solubilized; here, phospholipids and FAs form a bilayer in the interior and the bile salts occupy the edge, rendering it hydrophilic Released FAs and monoacylglycerols are incorporated into bile acids micelles Micelles move lipids from the intestinal lumen to the cell surface where absorption occurs (by diffusion through the plasma membrane) Micelles also serve as transport vehicles for vitamins A, K Fat malabsorption can result from pancreatic failure or lack of bile acids bulk of unabsorbed lipids is excreted with the stool…steatorrhea Fat digestion and absorption Most absorbed lipids are incorporated into chylomicrons Within the intestinal cell (after absorption): FAs of medium chain lenght (6-10C) pass through the cell into the portal blood without modification Long-chain FAs (> 12C) are bound to a fatty acid binding protein in the cytoplasm and transported to ER, where they are resynthesized into TGs • TGs form lipid globules to which phospholipids, cholesterol (esters), and apolipoproteins adsorb – chylomicrons • chylomicrons migrate through the Golgi to the basolateral membrane, are released and pass into the lymphatics DIGESTION OF NUCLEIC ACIDS In pancreas, dietary nucleic acids are hydrolyzed by: ribonucleases deoxyribonucleases endo- as well as exonucleases In the small intestine, polynucleotidases complete the hydrolysis to nucleotides which are then hydrolyzed to nucleosides by phosphatases and nucleotidases Nucleosides are directly absorbed into enterocyte or undergo further degradation by nucleosidases (nucleoside phosphorylases) to free bases and pentose-1-phosphate pyrimidine nucleosides are catabolized to -alanine/-aminoisobutyrate; or, they can be absorbed intact and utilized for the resynthesis of nucleic acids purine nucleosides are catabolized to uric acid; alternatively, purines are released and used for resynthesis of NA