Digestive enzymes

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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

…and there are 3 compartments where digestion and absorption occur:  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

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 A 2 cell membranes converts lecithin to lysolecithin that can damage

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 lumen of the GIT: in the       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

chymotrypsinogen, proelastase, procarboxypeptidases, prophospholipase enteropeptidase trypsinogen (produced in duodenum) trypsin – 6 N-terminal AA autocatalytic activation 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

Organ

Salivary gland

Secretion

NaCl, amylase

Secretagogue

acetylcholine Stomach HCl, pepsinogen Pancreas NaCl, enzymes NaHCO 3 , NaCl acetylcholine, histamine, gastrin (peptide) acetylcholine, cholecystokinin secretin   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

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 – N-terminus – carboxypeptidases aminopeptidases

Classes of peptidases

Type

Serine proteases Cysteine proteases Aspartate proteases Metalloproteases

Active site Ser

, His, Asp

Cys

, His 2 x Asp Zn 2+ (coordinated to AA)

pH optimum

7-9 3-6 2-5 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 pancreatic NaHCO 3  depend on neutralization of gastric HCl by  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  maltose, maltotriose,  -limit dextrins products:

 2) Hydrolysis of

oligosaccharides

intestinal epithelial cells – is carried out by surface enzymes of the 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 H 2 , CH 4 , CO 2 , H 2 S, 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  lactose  accumulation and bacterial fermentation of 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  surfactants : stabilize the water-lipid interface  act as 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 A 2 for activity): (requires bile acids   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 (H 2 N-CH 2 CH 2 SO 3 )  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 endo- as well as exonucleases  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

 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 NH 4 + , CO 2 , 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