Transcript Digestive enzymes - Univerzita Karlova v Praze

Digestive enzymes
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From food intake to absorption
 Mechanical homogenization of food, mixing with fluids secreted by the
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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
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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:
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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):
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endopeptidases – attack internal bonds:
• pepsin
• trypsin
• chymotrypsin
• elastase
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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)
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-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
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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:
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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:
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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
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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
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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):
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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:
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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
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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