1. Gastro-intestinal Enzymes

2. Pepsin Proteolytic enzyme of the stomach Proteolytic enzyme of the stomach At least two immunologically distinct pepsinogens (1 and 2) are secreted by the stomach At least two immunologically distinct pepsinogens (1 and 2) are secreted by the stomach Pepsinogen 1 is secreted by the chief cells in the oxyntic glandular area Pepsinogen 1 is secreted by the chief cells in the oxyntic glandular area Pepsinogen 2 by cells through out the stomach as well as in Brunner ’ s glands in the duodenum Pepsinogen 2 by cells through out the stomach as well as in Brunner ’ s glands in the duodenum It is an endopeptidase It is an endopeptidase It preferentially hydrolyses peptide linkage where one of the amino acids is aromatic (eg, tyrosin) or dicarboxylic amino acids (eg, glutamate) It preferentially hydrolyses peptide linkage where one of the amino acids is aromatic (eg, tyrosin) or dicarboxylic amino acids (eg, glutamate) Pepsin potentiates rather than initiates ulcer formation Pepsin potentiates rather than initiates ulcer formation

3. Clinical Significance Serum concentration of pepsinogen I reflects the parietal cell mass and correlate well with the maximum acid- secreting capacity Serum concentration of pepsinogen I reflects the parietal cell mass and correlate well with the maximum acid- secreting capacity Increased pepsinogen I: Increased pepsinogen I: – Duodenal ulcer – Acute and chronic superficial gastritis. – H. pylori sero-positive patients Decreased levels of pepsinogen I: Decreased levels of pepsinogen I: – Atrophic gastritis – Gastric carcinoma Increased pepsinogen II: Increased pepsinogen II: – Acute and chronic superficial gastritis Major risk factor for gastric ulcer Major risk factor for gastric ulcer

4. Pancreatic Juice Alkaline secretion: secreted by the duct cells Alkaline secretion: secreted by the duct cells – Composition: HCO 3 -, Na +, K +, Mg 2 +, Ca 2 +, Cl - – Function: together with the other alkaline secretions (bile and intestinal juice) neutralize the acid chyme arriving from the stomach. Why this is important? The pancreatic enzymes require a neutral or slightly alkaline pH for their activity The pancreatic enzymes require a neutral or slightly alkaline pH for their activity The absorption of fat depends on the formation of micelles, a process which only takes place at neutral or slightly alkaline pH The absorption of fat depends on the formation of micelles, a process which only takes place at neutral or slightly alkaline pH It protects the intestinal mucosa, excess acid in the duodenum can damage the mucosa and can lead to ulcer formation It protects the intestinal mucosa, excess acid in the duodenum can damage the mucosa and can lead to ulcer formation

5. Pancreatic Juice Pancreatic enzymes: secreted by acinar cells Pancreatic enzymes: secreted by acinar cells – In their inactive form; Trypsinogen Trypsinogen Chymotrypsinogen Chymotrypsinogen Proelastase Proelastase Procarboxypeptidase Procarboxypeptidase Prophospholipase A 2 Prophospholipase A 2 – In their active form; Lipase Lipase Alpha-amylase Alpha-amylase Ribonuclease Ribonuclease Deoxyribonuclease Deoxyribonuclease Cholesterol esterase Cholesterol esterase

7. Trypsin Two types, trypsinogen-1 and -2 Two types, trypsinogen-1 and -2 Trypsin is a serine proteinase charaterized by the presence at the active site of serine and histidine, both of which participate in the catalytic process Trypsin is a serine proteinase charaterized by the presence at the active site of serine and histidine, both of which participate in the catalytic process Trypsins are considered endopeptidases Trypsins are considered endopeptidasesendopeptidases The enzymatic mechanism is like all other serine proteases: A catalytic triad serves to make the active site serine nucleophilic. This is achieved by modifying the electrostatic environment of the serine residue The enzymatic mechanism is like all other serine proteases: A catalytic triad serves to make the active site serine nucleophilic. This is achieved by modifying the electrostatic environment of the serine residue catalytic triadactive siteserine catalytic triadactive siteserine The aspartate residue (Asp 189) located in the catalytic pocket (S1) of trypsins is responsible for attracting and stabilizing positively-charged lysine and/or arginine, and is thus responsible for the specificity of the enzyme. The aspartate residue (Asp 189) located in the catalytic pocket (S1) of trypsins is responsible for attracting and stabilizing positively-charged lysine and/or arginine, and is thus responsible for the specificity of the enzyme.aspartatelysinearginineaspartatelysinearginine

8. Trypsin Trypsin activity is stimulated by calcium and magnesium ions and to a lesser extent by cobalt and manganese ions. Cyanide, sulfide, citrate, fluoride, and heavy metals inhibit activity as do those organic phosphorous compounds that combine with serine at the active site. Trypsin activity is stimulated by calcium and magnesium ions and to a lesser extent by cobalt and manganese ions. Cyanide, sulfide, citrate, fluoride, and heavy metals inhibit activity as do those organic phosphorous compounds that combine with serine at the active site. Clinical Significance: Clinical Significance: – deficiency of trypsin might lead to the disorder termed meconium ileus meconium ileus meconium ileus – After an attack of acute pancreatitis, serum immunoreative trypsin rises in parallel with serum amylase activity.

9. Chymotrypsin Two types, chymotrypsinogen-1 and -2 Two types, chymotrypsinogen-1 and -2chymotrypsinogen Chymotrypsin is synthesized in the acinar cells of the human pancreas Chymotrypsin is synthesized in the acinar cells of the human pancreaspancreas Chymotrypsin-1 describes as anionic and chymotrypsin-2 as cationic because of their differing electrophoretic mobilities; the cationic form predominates. Chymotrypsin-1 describes as anionic and chymotrypsin-2 as cationic because of their differing electrophoretic mobilities; the cationic form predominates.

10. Action and kinetics of chymotrypsin: For an enzyme-mediated reaction to take place, the reacting molecule or molecules, called substrates, must fit into a specific section of the enzymes structure called the active site. For an enzyme-mediated reaction to take place, the reacting molecule or molecules, called substrates, must fit into a specific section of the enzymes structure called the active site. Each active site has: Each active site has: (1) a shape that fits a specific substrate or substrates (2) side chains that attract the enzymes particular substrates (3) side chains specifically positioned to speed the reaction.

11. Mechanism of peptide bond cleavage in chymotrypsin (1)

12. Mechanism of peptide bond cleavage in chymotrypsin (2)

13. Mechanism of peptide bond cleavage in chymotrypsin (3)

14. Mechanism of peptide bond cleavage in chymotrypsin (4)

15. Mechanism of peptide bond cleavage in chymotrypsin (5)

16. Mechanism of peptide bond cleavage in chymotrypsin (6)

17. Chymotrypsin Clinical Significance: Clinical Significance: – Chymotrypsin is more resistant than trypsin to degradation in the intestine; it is therefore the enzyme of choice for assay in feces. – The major application of chymotrypsin assay is the investigation of pancreatic disease.

18. Acute Pancreatitis Acute pancreatitis is a disease in which the pancreatic tissue is destroyed by digestive enzymes Acute pancreatitis is a disease in which the pancreatic tissue is destroyed by digestive enzymes The pancreas normally secrets a polypeptide known as Kazal inhibitor, that inhibits any small amounts of activated trypsin which may find its way into the ducts, by forming a complex with it The pancreas normally secrets a polypeptide known as Kazal inhibitor, that inhibits any small amounts of activated trypsin which may find its way into the ducts, by forming a complex with it Enzyme Y is also exhibiting a protective function, which is activated by traces of active trypsin degrades zymogen Enzyme Y is also exhibiting a protective function, which is activated by traces of active trypsin degrades zymogen The alkaline pH (8.0-9.5) and low Ca 2+ concentration in pancreatic secretions promote the degradation rather than the activation of trypsinogen The alkaline pH (8.0-9.5) and low Ca 2+ concentration in pancreatic secretions promote the degradation rather than the activation of trypsinogen In acute pancreatitis activated trypsin and other enzymes are present in the ducts of the pancreas In acute pancreatitis activated trypsin and other enzymes are present in the ducts of the pancreas

19. Elastase Elastase, like trypsin and chymotrypsin, is a serine protease that also hydrolyses amides and esters Elastase, like trypsin and chymotrypsin, is a serine protease that also hydrolyses amides and esters Attacking bonds next to small amino acids residues such as glycine, alanine and serine Attacking bonds next to small amino acids residues such as glycine, alanine and serine Two types; Two types; – Elastase I, which is anionic exists in serum both free and as a complex with alpha-1 proteinase inhibitor; – Elastase II which is cationic mainly in the bound form with alpha-1 proteinase inhibitor

20. Elastase Clinical Significance: Clinical Significance: – Elastase I is increased in acute and relapsing chronic pancreatitis – There is some evidence that this enzyme is considerably more specific for pancreatitis than is amylase. – Elevation of elastase I have also been observed in carcinoma of the pancreas, especially carcinoma of the head of the pancreas. – Elastase II is likewise increased in acute pacreatitis

21. Enteropeptidase: Enteropeptidase: – Is a serine protease enzyme secreted by intestinal mucusa serine proteaseenzymeserine proteaseenzyme – Cleaves after Lysine if the Lys is preceded by four Asp and not followed by a Pro. Carboxypeptidase: Carboxypeptidase: – Carboxypeptidases are usually classified into one of several families based on their active site mechanism Enzymes that use a metal in the active site are called "metallo- carboxypeptidases". Enzymes that use a metal in the active site are called "metallo- carboxypeptidases". Carboxypeptidases that use active site serine residues are called "serine carboxypeptidases". Carboxypeptidases that use active site serine residues are called "serine carboxypeptidases".serine Carboxypeptidases that use an active site cysteine are called "cysteine carboxypeptidase" (or "thiol carboxypeptidases"). Carboxypeptidases that use an active site cysteine are called "cysteine carboxypeptidase" (or "thiol carboxypeptidases").cysteinethiolcysteinethiol – By substrate preference: In this classification system, carboxypeptidases that have a stronger preference for those amino acids containing aromatic or branched hydrocarbon chains are called carboxypeptidase A (A for aromatic/aliphatic). In this classification system, carboxypeptidases that have a stronger preference for those amino acids containing aromatic or branched hydrocarbon chains are called carboxypeptidase A (A for aromatic/aliphatic).aromaticbranchedcarboxypeptidase Aaliphaticaromaticbranchedcarboxypeptidase Aaliphatic Carboxypeptidases that cleave positively charged amino acids (arginine, lysine) are called carboxypeptidase B (B for basic). Carboxypeptidases that cleave positively charged amino acids (arginine, lysine) are called carboxypeptidase B (B for basic).argininelysinecarboxypeptidase Bbasicargininelysinecarboxypeptidase Bbasic

22. Induced conformation ( carboxypeptidase A)

23. Brush Border Enzymes of The Intestine Aminopeptidase, Leucine aminopeptidase Aminopeptidase, Leucine aminopeptidase Oligopeptidases, degrade small peptides such as tetrapeptides Oligopeptidases, degrade small peptides such as tetrapeptides Dipeptidyl aminopeptidases, remove dipeptides from the N-terminal of proteins Dipeptidyl aminopeptidases, remove dipeptides from the N-terminal of proteins Tripeptidases and Dipeptidases in the endothelial cells Tripeptidases and Dipeptidases in the endothelial cells

24. Lipases Human lipase is a glycoprotien Human lipase is a glycoprotien Lipases are defined as enzymes that hydrolyze glycerol esters of long-chain fatty acids. Lipases are defined as enzymes that hydrolyze glycerol esters of long-chain fatty acids. For full catalytic activity and greatest specificity, the presence of bile salts and a cofactor, called co-lipase is required. For full catalytic activity and greatest specificity, the presence of bile salts and a cofactor, called co-lipase is required. Both lipase and colipase are synthesized in the pancreatic acinar cells and secreted by the pancreas in roughly equimolar quantities. Both lipase and colipase are synthesized in the pancreatic acinar cells and secreted by the pancreas in roughly equimolar quantities. Lipase measurements are used exclusively to investigate pancreatic disorders, usually pancreatitis Lipase measurements are used exclusively to investigate pancreatic disorders, usually pancreatitis

26. Brush Border Enzymes of The Intestine Isomaltase (alpha 1,4-glycosidase) Isomaltase (alpha 1,4-glycosidase) Glucoamylase Glucoamylase Maltase Maltase Sucrase Sucrase Lactase Lactase Trehalase Trehalase Sucrase and isomaltase are synthesized as a single polypeptide chain inside the cell Sucrase and isomaltase are synthesized as a single polypeptide chain inside the cell

27. Lactase Secreted by Brunner's glands of the duodenum and glands of Lieberkun, works at pH 5.4-6.0 hydrolyze lactose to glucose and galactose. Secreted by Brunner's glands of the duodenum and glands of Lieberkun, works at pH 5.4-6.0 hydrolyze lactose to glucose and galactose. Lactase deficiency: Lactase deficiency: – Symptoms of flatulence, abdominal discomfort, bloating, or diarrhea Lactase deficiency of two types: Lactase deficiency of two types: – Congenital Lactase Deficiency Symptoms occur as soon as milk is taken Symptoms occur as soon as milk is taken Absent or low intestinal lactase in the neonate, however, cannot be taken as proof of congenital deficiency because lactase is normally the slowest of the oligosaccharidases to reach normal levels in the newborn’s gut Absent or low intestinal lactase in the neonate, however, cannot be taken as proof of congenital deficiency because lactase is normally the slowest of the oligosaccharidases to reach normal levels in the newborn’s gut An abnormal oral lactose tolerance test obtained a few months after birth could also be due to congenital glucose-galactose intolerance An abnormal oral lactose tolerance test obtained a few months after birth could also be due to congenital glucose-galactose intolerance

28. Lactase Acquired Lactase Deficiency Acquired Lactase Deficiency – Lactase activity declines as a child ages – The age at which decline begins is genetically determined in an Autosomal recessive fashion and differs among ethnic groups Secondary Lactase Intolerance Secondary Lactase Intolerance – Occurs as a result of reduced enzyme activity following diffuse intestinal damage from; – Infections (Giardiasis, bacterial overgrowth, or viruses), – Ulcerative colitis, – Celiac disease, and – Tropical sprue Testing: hydrogen breath test is used to measure the amount of hydrogen in the breath. Testing: hydrogen breath test is used to measure the amount of hydrogen in the breath.

29. Ribonuclease (RNase): digestion of dietary nucleic acids Ribonuclease (RNase): digestion of dietary nucleic acids – Ribonuclease A catalyzes cleavage of the phosphodiester bond between the 5'-ribose of a nucleotide and the phosphate group attached to the 3'-ribose of an adjacent pyrimidine nucleotide forming a 2',3'-cyclic phosphate – Ribonuclease B: is a glycosylated derivative of RNase A. De oxyribonuclease (DNase): digestion of dietary nucleic acid De oxyribonuclease (DNase): digestion of dietary nucleic acid Polynucleotidases: nucleic acids into nucleotides Polynucleotidases: nucleic acids into nucleotides Nucleosidases: nucleosides to give free nitrogen base plus pentose phosphate Nucleosidases: nucleosides to give free nitrogen base plus pentose phosphate Phosphatases: remove phosphate Phosphatases: remove phosphate Phospholipases: phospholipids to produce glycerol, fatty acids, phosphoric acid, and bases such as choline Phospholipases: phospholipids to produce glycerol, fatty acids, phosphoric acid, and bases such as choline Cholesterol esterase: cholesterol and fatty acids Cholesterol esterase: cholesterol and fatty acids

30. Sucrase-Isomaltase deficiency: flatulence, abdominal discomfort, bloating, diarrhea Sucrase-Isomaltase deficiency: flatulence, abdominal discomfort, bloating, diarrhea Colipase deficiency: steatorrhea Colipase deficiency: steatorrhea Absorption of undigested polypeptides may cause antigenic reactions: celiac disease Absorption of undigested polypeptides may cause antigenic reactions: celiac disease Monosaccharide Malabsorption: caused by single mutation resulted in a defect in the Na + -glucose co-transporter carrier mechanism (SGLT1) Monosaccharide Malabsorption: caused by single mutation resulted in a defect in the Na + -glucose co-transporter carrier mechanism (SGLT1)

31. Disturbances due to malabsorption Anemia: Iron, Vitamin B 12, Folate Anemia: Iron, Vitamin B 12, Folate Edema: Products of protein digestion Edema: Products of protein digestion Tetany: Calcium, Magnesium, Vitamin D Tetany: Calcium, Magnesium, Vitamin D Osteoporosis: Calcium, Product of protein digestion, Vitamin D Osteoporosis: Calcium, Product of protein digestion, Vitamin D Milk intolerance: Lactose Milk intolerance: Lactose Bleeding Bruising: Vitamin K Bleeding Bruising: Vitamin K Steatorrhea (fatty stools): Lipids and fat-soluble vitamins Steatorrhea (fatty stools): Lipids and fat-soluble vitamins Hartnup disease (defect in intestinal neutral amino acid carrier): Neutral amino acids Hartnup disease (defect in intestinal neutral amino acid carrier): Neutral amino acids