BIOCHEMISTRY: A TEXTBOOK FOR UNIVERSITIES - E. S. Severin - 2004

CHAPTER 9. METABOLISM AND FUNCTIONS OF AMINO ACIDS

III. Protein Digestion

The content of free Amino Acids in food products is extremely low. The vast majority of them are incorporated into Proteins, which are hydrolyzed in the gastrointestinal tract (GIT) by protease Enzymes (peptidases). The substrate Specificity of these enzymes lies in the fact that each of them most rapidly cleaves peptide bonds formed by specific amino acids. Proteases that hydrolyze peptide bonds within the protein molecule are classified as Endopeptidases. Enzymes belonging to the exopeptidase group hydrolyze peptide bonds formed by terminal amino acids. Under the action of all GIT proteases, dietary proteins are broken down into individual amino acids, which are subsequently absorbed into tissue Cells.

A. Protein Digestion in The Stomach

Gastric juice is a product of several types of cells. Parietal cells in the gastric walls produce Hydrochloric acid, while chief cells secrete pepsinogen. Mucous neck cells and other gastric epithelial cells secrete mucin-containing mucus. Parietal cells also secrete a glycoprotein known as the "intrinsic factor" (Castle's factor) into the gastric lumen. This protein binds the "extrinsic factor" — vitamin B₁₂, preventing its degradation and facilitating its absorption.

1. Formation and Role of Hydrochloric Acid

The primary digestive function of the stomach is to initiate protein digestion, a process in which hydrochloric acid plays a crucial role. Proteins entering the stomach stimulate the release of histamine and a group of protein Hormones known as gastrins (see Chapter 11), which in turn trigger the secretion of HCl and the proenzyme pepsinogen. HCl is produced in the parietal cells of gastric glands through the reactions illustrated in Fig. 9-2.

Class="center">Fig. 9-2. Secretion of hydrochloric acid in the stomach. 1 — Carbonic anhydrase; 2 — H⁺/K⁺-ATPase; 3 — anion exchanger proteins; 4 — chloride channel.

The source of H⁺ is H₂CO₃, which is formed within the gastric parietal cells from CO₂ diffusing from the Blood and H₂O under the action of the enzyme carbonic anhydrase (carbonate dehydratase):

H₂O + CO₂ —> H₂CO₃ —> HCO₃^- + H⁺.

The dissociation of H₂CO₃ yields bicarbonate, which is transported into the plasma in exchange for Cl^- via specific transport proteins, and H⁺ ions, which enter the gastric lumen via Active Transport catalyzed by membrane-bound H⁺/K⁺-ATPase. This process increases the proton concentration in the gastric lumen by 10⁶-fold. Cl^- ions enter the gastric lumen through a chloride channel.

The concentration of HCl in gastric juice can reach 0.16 M, reducing the pH to 1.0 — 2.0. The ingestion of protein-rich meals is often followed by The excretion of alkaline urine due to the secretion of large amounts of bicarbonate during HCl production.

HCl induces the Denaturation of uncooked dietary proteins, thereby increasing the accessibility of peptide bonds to proteases. Furthermore, HCl exerts a bactericidal effect, preventing pathogenic Bacteria from entering the intestine. In addition, hydrochloric acid activates pepsinogen and establishes the optimal pH for Pepsin activity.

2. Mechanism of Pepsin Activation

Stimulated by gastrins, the chief Cells of the gastric glands synthesize and secrete pepsinogen, the inactive precursor of pepsin. Pepsinogen is a single-polypeptide protein with a Molecular Weight of 40 kDa. Upon exposure to HCl, it is converted into active pepsin (molecular weight 32.7 kDa) with an optimal pH of 1.0 — 2.5. During activation, partial proteolysis cleaves 42 amino acid residues from the N-terminus of the pepsinogen molecule, which include nearly all of the positively charged amino acids present in pepsinogen. Consequently, negatively charged amino acids predominate in active pepsin, playing a key role in molecular conformational changes and The formation of the Active Site. The active pepsin molecules generated by HCl action rapidly activate the remaining pepsinogen molecules (autocatalysis). Pepsin primarily hydrolyzes peptide bonds in proteins formed by aromatic amino acids (phenylalanine, Tryptophan, Tyrosine) and, somewhat more slowly, those formed by leucine and dicarboxylic amino acids. Since pepsin is an endopeptidase, its action in the stomach yields shorter Peptides rather than free amino acids.

3. Age-Related Features of protein DIGESTION IN THE Stomach

In infants, the stomach contains the enzyme rennin (chymosin), which causes milk to curdle. The main milk protein is casein, a mixture of several proteins that differ in Amino Acid Composition and electrophoretic mobility. Rennin catalyzes the Cleavage of a glycopeptide from casein, resulting in the formation of paracasein. Paracasein binds Ca²⁺ ions to form an insoluble clot, which prevents milk from leaving the stomach too rapidly, allowing proteins sufficient time to be digested by pepsin. Adult stomachs lack rennin; in adults, milk is curdled through the combined action of HCl and pepsin.

Another protease, gastricsin, has been identified in the human gastric mucosa. All three enzymes (pepsin, rennin, and gastricsin) share similar primary structures, indicating that they evolved from a common ancestral Gene.

4. Disorders of protein Digestion in the Stomach

Various gastrointestinal disorders impair the gastric secretion of HCl and pepsinogen, leading to a marked decrease in protein digestion. Pathological alterations in gastric juice acidity are the most common. Impaired pepsin production is observed less frequently and is associated with more severe gastric damage.

Determining gastric juice acidity is used to diagnose various gastric diseases (Table 9-2). Hyperacidity of gastric juice is typically accompanied by heartburn and diarrhea, and can be a symptom of gastric and duodenal ulcers, as well as hyperacid gastritis. Hypoacidity occurs in certain types of gastritis. Complete absence of HCl and pepsin (gastric achylia) is observed in atrophic gastritis and is frequently accompanied by pernicious anemia due to deficient production of Castle's factor and impaired vitamin B₁₂ absorption (see Chapter 3). Anacidity (gastric juice pH > 6.0) indicates significant loss of hydrochloric acid-secreting parietal cells in the gastric mucosa, which is often a precursor to Gastric Cancer.

Table 9-2. Components of gastric juice in health and pathological states

The acidity of gastric juice is expressed in titration units (TU), defined as the volume (in milliliters) of 0.1 M NaOH required to titrate 100 ml of gastric juice using a specific indicator. When determining the acidity of gastric juice, researchers distinguish between total acidity, bound HCl, and free HCl.

Total acidity of gastric juice — the sum of all acid-reacting substances in gastric juice, representing the gastric secretion collected over a 1-hour period. Normal values for total acidity range from 40 to 60 TU.

Bound hydrochloric acid — HCl bound to proteins and their digestion products. Normal values of bound HCl in healthy individuals range from 20 to 30 TU.

Free HCl — hydrochloric acid not bound to Other components of gastric juice. Normal values of free HCl range from 20 to 40 TU.

Normal pH of gastric juice ranges from 1.5 to 2.0.

Lactic acid is normally absent in gastric juice. It appears when the concentration of free hydrochloric acid is reduced or absent due to the proliferation of lactic acid bacteria, or in cases of malignant gastric tumors where glucose is oxidized anaerobically.

In the Diagnosis of gastric diseases, biochemical analyses are complemented by mandatory X-ray and endoscopic examinations, as well as biopsy.

B. Protein Digestion in the Intestine

During digestion, the gastric contents (chyme) pass into the duodenum. The low pH of the chyme stimulates the release of the protein hormone secretin into the bloodstream. This hormone, in turn, triggers the Pancreas to secrete pancreatic juice containing HCO₃^- into the Small Intestine, neutralizing gastric HCl and inhibiting pepsin. As a result, the pH sharply rises from 1.5–2.0 to ~7.0.

The entry of peptides into the small intestine stimulates the secretion of another protein hormone, cholecystokinin (see Chapter 11), which promotes the release of pancreatic enzymes with an optimal pH of 7.5–8.0. Protein digestion is completed through the combined action of Pancreatic and Intestinal enzymes.

1. Activation of Pancreatic Enzymes

The pancreas synthesizes a series of protease zymogens: trypsinogen, chymotrypsinogen, proelastase, and procarboxypeptidases A and B. In the intestine, these precursors are converted into active enzymes—Trypsin, Chymotrypsin, Elastase, and Carboxypeptidases A and B—via Limited proteolysis.

Trypsinogen activation is triggered by enteropeptidase, an enzyme located in the intestinal epithelium. This enzyme cleaves a hexapeptide, Val-(Asp)₄-Lys, from the N-terminus of the trypsinogen molecule. Conformational Changes in the remaining polypeptide chain form the active site, yielding active trypsin. The Val-(Asp)₄-Lys sequence is conserved across most known trypsinogens from various organisms, ranging from fish to humans.

The resulting trypsin activates chymotrypsinogen, producing several active enzymatic forms (Fig. 9-3). Chymotrypsinogen consists of a single polypeptide chain comprising 245 amino acid residues and five disulfide bridges. Trypsin catalyzes the Cleavage of the peptide bond between amino acids 15 and 16, yielding active π-chymotrypsin. Subsequently, π-chymotrypsin cleaves the dipeptide Ser(14)-Arg(15), forming δ-chymotrypsin. The cleavage of the Thr(147)-Arg(148) dipeptide completes the Formation of the stable active enzyme, α-chymotrypsin, which consists of three polypeptide chains linked by disulfide bridges.

Fig. 9-3. Activation of chymotrypsinogen. The chymotrypsinogen molecule consists of 245 amino acid residues and contains five disulfide bridges. The diagram highlights the sites of proteolytic cleavage. α-Chymotrypsin, the stable active form of the enzyme, is composed of three polypeptide chains covalently linked by two Disulfide Bonds and non-covalently stabilized by Hydrogen Bonds and hydrophobic interactions.

The remaining pancreatic protease zymogens (proelastase and procarboxypeptidases A and B) are similarly activated by trypsin through limited proteolysis, generating the active enzymes elastase and carboxypeptidases A and B.

2. Enzyme Specificity of Proteases

Trypsin preferentially hydrolyzes peptide bonds formed by the carboxyl groups of Arginine and Lysine. Chymotrypsins exhibit the highest activity toward peptide bonds formed by the carboxyl groups of aromatic amino acids (Phe, Tyr, Trp).

Carboxypeptidases A and B are zinc-containing enzymes that cleave C-terminal amino acid residues. Specifically, Carboxypeptidase A preferentially releases amino acids with aromatic or hydrophobic side chains, whereas carboxypeptidase B targets arginine and lysine residues.

The final stage of digestion involves the Hydrolysis of small peptides by aminopeptidases and dipeptidases, which are synthesized in their active forms by the cells of the small intestine.

Aminopeptidases sequentially cleave N-terminal amino acids from the peptide chain. The best-known example is leucine aminopeptidase—a Zn²⁺- or Mn²⁺-containing enzyme that, despite its name, displays broad specificity toward various N-terminal amino acids.

Dipeptidases cleave dipeptidases into free amino acids but have no activity against tripeptidases.

As a result of the sequential action of all digestive proteases, the majority of dietary proteins are broken down into free amino acids.

C. Cellular Protection Against Proteases

The cells of the pancreas are protected from the action of digestive enzymes because:

✵ these enzymes are produced as inactive precursors within the pancreatic cells and are activated only after secretion into the intestinal lumen. Thus, the site of synthesis and The Site of Action of these enzymes are spatially separated.

✵ the pancreatic cells contain a trypsin inhibitor protein, which forms a stable complex with the active form of the enzyme in the event of premature activation.

In the stomach and intestinal lumen, proteases do not come into contact with cellular proteins because the mucosa is covered with a layer of mucus, and every Cell bears Polysaccharides on the outer surface of its Plasma Membrane that are resistant to protease cleavage, thereby protecting The Cell from their action.

Destruction of cellular proteins by proteases occurs in PEPTIC ULCER DISEASE of the stomach or duodenum. However, the initial mechanisms of ulcer formation remain poorly understood.

G. Transport of Amino Acids into Cells

Amino acids resulting from protein digestion are rapidly absorbed in the intestine. Their transport occurs via two pathways: through the hepatic portal system, leading directly to the Liver, and via Lymphatic vessels, which communicate with the blood through the thoracic lymphatic duct. Peak blood concentrations of Amino acids are reached 30–50 min after the ingestion of protein-rich food (CARBOHYDRATES and fats delay amino acid absorption). The absorption of L-amino acids (but not D-isomers) is an active process requiring Energy Expenditure. Amino acids are transported across the intestinal wall from its mucosal surface into the blood (Fig. 9-4). Passage across the brush border is mediated by a variety of transporters, many of which operate via Na⁺-dependent symport mechanisms, similar to glucose transport (see Section 7).

Fig. 9-4. Mechanism of amino acid Absorption in the intestine. An L-amino acid enters the enterocyte via symport with an Na⁺ ion. Subsequently, a specific translocase carries The amino acid across the membrane into the bloodstream. The exchange of sodium ions across cellular membranes is driven by primary active transport via Na⁺, K⁺-ATPase.

Variations in The rate of amino acid penetration across cell membranes indicate the existence of transport systems that facilitate amino acid transfer through both the outer plasma membrane and intracellular membranes. Currently, at least five specific transport systems are known, each functioning to transfer a specific group of structurally related amino acids:

✵ neutral amino acids with a short side chain (Alanine, Serine, Threonine);

✵ neutral amino acids with a long or branched side chain (valine, leucine, isoleucine);

✵ amino acids with cationic side chains (lysine, arginine);

✵ amino acids with anionic side chains (glutamic and aspartic acids);

✵ imino acids (Proline, hydroxyproline).

Notably, Na⁺-dependent carriers include transporters for amino acids of the first and fifth groups, as well as the Methionine transporter. Na⁺-independent carriers are specific for certain neutral amino acids (phenylalanine, leucine) and amino acids with cationic side chains (lysine).

Amino acids compete with one another for specific binding sites. For example, the absorption of leucine (if present at a sufficiently high concentration) reduces the uptake of isoleucine and valine.

One of the specific transport systems for certain neutral amino acids operates in the intestine, Kidneys, and presumably the Brain. It is known as the y-glutamyl cycle (Fig. 9-5).

Fig. 9-5. The y-glutamyl cycle. The system consists of one membrane-bound and five cytoplasmic enzymes. The transfer of an amino acid into the cell is carried out in a complex with the glutamyl residue of Glutathione, mediated by y-glutamyl transferase. The amino acid is subsequently released, while the y-glutamyl residue undergoes several enzymatic steps to be converted back into glutathione, which can then accept another amino acid molecule. E₁ — y-glutamyl transferase; E₂ — y-glutamylcyclotransferase; E₃ — peptidase; E₄ — oxoprolinase; E₅ — y-glutamylcysteine synthetase; E₆ — glutathione synthetase.

This system involves 6 enzymes, one of which is located in The cell membrane, while the others reside in the Cytosol. A pivotal role in Amino Acid Transport is played by the membrane-bound enzyme y-glutamyl transferase. This glycoprotein catalyzes the transfer of the y-glutamyl group from glutathione (or occasionally another y-glutamyl peptide) to the amino acid being transported, followed by translocation of the complex into the cell. Glutathione is a tripeptide—y-glutamylcysteinylglycine—found in all animal Tissues. The reaction proceeds as outlined below (see Scheme A on p. 468).

The amino acid, bound to the y-glutamyl residue, ends up inside the cell. The next reaction involves the cleavage of the y-glutamyl residue by the action of the enzyme y-glutamylcyclotransferase (see Scheme B).

The dipeptide cysteinylglycine is hydrolyzed by a peptidase into Two amino acids: Cysteine and Glycine. As a result of these three reactions, a single amino acid molecule is transported into the cell (or intracellular Structure). The subsequent three reactions ensure the regeneration of glutathione, allowing the cycle to repeat continuously. The transport of a single amino acid molecule via the y-glutamyl cycle requires the expenditure of 3 ATP molecules.

D. Disorders of Protein Digestion and Amino Acid Transport

A small fraction of protein digestion products consists of non-hydrolyzed short peptides. Some individuals develop an immune reaction to protein ingestion, which is clearly associated with The ability to absorb such peptides. The products of fully digested protein (amino acids) lack antigenic properties and do not trigger immune responses.

In newborns, the permeability of the intestinal mucosa is higher than in adults; therefore, Antibodies from colostrum (the secretion of Mammary Glands produced in the first days after birth, enriched with antibodies and antitoxins) can enter the bloodstream. This is exacerbated by the presence of a trypsin inhibitor protein in colostrum. Proteolytic Enzymes in the digestive secretions of newborns have low activity. All of this facilitates the intestinal absorption of small amounts of native proteins sufficient to induce an Immune Response. Evidently, such enhanced intestinal absorptive capacity accounts for the occasional intolerance to dietary proteins (such as milk and eggs) observed in adults.

Increasing evidence Supports the hypothesis that celiac disease (nontropical sprue) involves Impairment of the intestinal mucosal cells where small non-hydrolyzed peptides are absorbed. Celiac disease is characterized by hypersensitivity to gluten, a storage protein found in cereal grains consumed in the human diet. This protein exerts a toxic effect on the small intestinal mucosa, leading to pathological changes and malabsorption. The Pathogenesis of the disease remains insufficiently understood.

Conditions such as cystinuria, Hartnup disease, and several others arise from defects in neutral amino acid transporters in the intestine and kidneys. A congenital pathology associated with a deficiency of the enzyme 5-oxoprolinase has also been described (Fig. 9-5, reaction 4), resulting in urinary excretion of oxoproline. These patients exhibit impaired amino acid transport into tissues and altered cellular METABOLISM.