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Transcript of Digestive enzymes [email protected].
Digestive enzymes
Various 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
Pancreatic enzymes together with bile are poured into the lumen of the descending part of the duodenum
Digestion of oligomers of AA and saccharides is accomplished by the enzymes in the luminal plasma membranes of enterocytes; these enzymes – usually glycoproteins
Hydrolysis of di- and tripeptides occurs in the cytoplasm of enterocytes
…and there are 3 compartments where digestion and absorption occur:
Zymogens
Digestive enzymes are usually synthesized as larger inactive precursors – zymogens
Otherwise they would digest the tissues that synthesize them:
acute pancreatitis: premature activation of digestive enzymes produced by pancreas → auto-digestion of pancreas; activated phospholipase A2 converts lecithin to lysolecithin that can damage cell membranes
Synthesis of zymogens
Proteins destined for secretion are synthesized on polysomes of the RER
Their N-terminus contains a signal sequence → release of the protein into ER; then, the signal sequence may be clipped off
Transport to the Golgi complex
The proteins are stored in vesicles; after stimulus, granules move to the luminal plasma membrane (PM) and fuse with PM …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 stomach cells
Pepsinogen is activated by the proteolytic removal of 44 AA from its N-terminus – either as an intramolecular reaction or by active pepsin
This reaction takes place at pH values below 5
Activation of pancreatic zymogens in the lumen of the small intestine
trypsinogen
enteropeptidase(produced in duodenum)
– 6 N-terminal AA
trypsin
autocatalytic activation
chymotrypsinogen, proelastase, procarboxypeptidases, prophospholipase
chymotrypsin, elastase, carboxypeptidases, phospholipase
„Strategies“ that prevent premature zymogen activation
At pH>2, the peptide (44 AA) clipped of pepsinogen remains bound to pepsin, masking its active site; it is released by a drop of pH below 2 or by further degradation by pepsin
Pancreatic secretory trypsin inhibitor (PSTI), 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
Cholecystokinin: peptide secreted by cells of small int. after stimulation by AA and peptides from gastric proteolysis, by FA, and by acid pH
Secretin: peptide secreted by cells of small int.; stimulated by luminal pH < 5
Organ Secretion Secretagogue
Salivary gland NaCl, amylase acetylcholine
Stomach HCl, pepsinogen acetylcholine, histamine, gastrin (peptide)
Pancreas NaCl, enzymes acetylcholine, cholecystokinin
NaHCO3, NaCl secretin
DIGESTION OF PROTEINS
By peptidases (proteases):
endopeptidases – attack internal bonds:• pepsin• trypsin• chymotrypsin• elastase
exopeptidases – cleave off 1 AA at a time from the:• C-terminus – carboxypeptidases• N-terminus – aminopeptidases
Classes of peptidases
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 AA) 7-9
Peptidases hydrolyze the peptide bond
…and differ in substrate specificity:
Pepsins
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 AA; 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 AA and small peptides (2-8 AA)
Intestinal peptidases
Luminal surface of intestinal epithelial cells contains endopeptidases, aminopeptidases, and dipeptidases that cleave oligopeptides released by pancreatic peptidases Products: AA, di- and tripeptides → absorbed by enterocytes
Di- and tripeptides are hydrolyzed by intestinal cytoplasmic peptidases AA are 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 products: maltose, maltotriose, -limit dextrins
2) Hydrolysis of oligosaccharides is carried out by surface enzymes of the intestinal epithelial cells – disaccharidases and oligosaccharidases
These enzymes – often exoglycosidases
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 celluloses, hemicelluloses, inulin, pectin, are resistant to human digestive enzymes
A small percentage of this „dietary fibre“ is hydrolyzed and then anaerobically metabolized by the bacteria of the lower intestinal tract
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
Inability to absorb lactose accumulation and bacterial fermentation of lactose production of gas (distension of gut, flatulence); osmotically active solutes 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
Steps in lipid digestion & absorption
Lipid digestion is initiated in stomach
In the stomach, acid-stable lipase, secreted by stomach (gastric lipase) and by lingual glands (lingual lipase), converts TG mostly into FA and 1,2-diacylglycerols (small amount of monoAG is also produced)
The products possess both polar and non-polar groups act as surfactants: stabilize the water-lipid interface dispersion of the lipid phase into smaller droplets (emulsification) better availability of the substrate to the lipases.
These lipases have the unique ability to initiate the degradation of maternal milk fat globules
Pancreatic lipase
Cleaves acylglycerols mainly to FA and 2-monoacylglycerols
Requires solubilization of the substrate
Also requires colipase (secreted by the pancreas) that anchors and activates the enzyme
Absorption of resulting FA and monoAG requires bile salts micelles
Digestion of phospholipids
By phospholipases, especially by phospholipase A2 (requires bile acids for activity):
FA 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
-)
A portion of the primary bile acids is subjected to the modifications by intestinal bacteria → secondary bile acids
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 the bile salt is oriented from the water molecules x hydrophilic region interacts with water
Mixed micelles contain (beside bile acids) phospholipids and cholesterol, or FA and acylglycerols; FA and phospholipids form a bilayer in the interior, bile salts occupy the edge.
Released FA and monoacylglycerols are incorporated into bile acids micelles
Micelles move lipids from the intestinal lumen to the cell surface where absorption occurs
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): FA of medium chain lenght (6-10C) pass into the portal blood without
modification
long-chain FA (> 12C) are bound to a fatty acid binding protein in the cytoplasm and transported to ER, where they are resynthesized to TG
• TG form lipid globules to which phospholipids, cholesterol (esters), and apolipoproteins adsorb – chylomicrons
• chylomicrons migrate through the Golgi to the basolateral membrane, they are released, and pass into the lymphatics
DIGESTION OF NUCLEIC ACIDS
Pancreatic enzymes hydrolyze dietary nucleic acids: ribonucleases deoxyribonucleases
Polynucleotidases of the small intestine complete the hydrolysis to nucleotides which are then hydrolyzed to nucleosides by phosphatases and nucleotidases
Nucleosides are used as such or undergo degradation by nucleosidases / nucleoside phosphorylases to free bases and pentose-1-phosphate
endo- as well as exonucleases
Purine nucleosides are: A) catabolized to uric acid B) alternatively, purines are released and used for resynthesis of NA
Pyrimidine nucleosides are:
A) catabolized to NH4+, CO2,
and β-aminoisobutyrate or β-alanine, respectively, that are partially converted to (methyl)malonyl-CoA
B) absorbed intact and utilized for the resynthesis of nucleic acids