Biochemistry of Proteins PDF

Biochemistry of proteins Proteins are macromolecules composed of polymers of covalently linked amino acids and that they are involved in every cellular process. Amino acids are not stored by the body Amino acids must be obtained from -Diet -Synthesized de novo, or...

Biochemistry of proteins Proteins are macromolecules composed of polymers of covalently linked amino acids and that they are involved in every cellular process. Amino acids are not stored by the body Amino acids must be obtained from -Diet -Synthesized de novo, or N -Produced from normal protein degradation. C H S O P ⦿At physiologic pH: The side chains of lysine & arginine are fully ionized and positively charged. 20 amino acids: 9 Essential Aas cannot be manufactured by the body & 11 non-essential AAs can be manufactured by the body with proper nutrition. Alanine Glutamic Acid Lysine Threonine Arginine* Glutamine Methionine Tryptophan Aspartic Acid Glycine Phenylalanine Tyrosine Asparagine Histidine* Proline Valine Cysteine-Cystine Isoleucine Serine Leucine  How to remember the essential amino acid? I Left Home To Make Isoleucine Leucine Histidine Tryptophan Methionine Visit Throught London Philipin Argantin Valine Threonine Lysine Phenylalanine Arginine Levels of Protein Structure A. Peptide bond (PB): In proteins, AAs are joined covalently by PB, which are amide linkages between the α-carboxyl group of one AA & the α-amino group of another. PBs are not broken by conditions that denature proteins (heating or high concentrations of urea). PBs are hydrolyzed by prolonged exposure to strong acid or base at elevated temperatures. Bonds contributing to tertiary structure D. Denaturation of proteins Protein denaturation results in the unfolding & disorganization of the protein's secondary & tertiary structures, which are not accompanied by hydrolysis of peptide bonds. Denatured proteins are often insoluble &, therefore, precipitate from solution. Denaturing agents Heat, Organic solvents Mechanical mixing Strong acids or bases Detergents Ions of heavy metals such as lead and mercury.  Overall protein metabolism Urea Fat Glycogen Body proteins NH3 Glucose Dietary Carbon CO2 + Amino acid pool ~ H 2O + proteins skeleton 100 g energy Ketone bodies Synthesis of non-essential amino acids ▪ Heme Fat, sterol ▪ Creatine ▪ Purines ▪ Pyrimidines ▪ Neurotransmitters ▪ Hormones ▪ Melanin ▪ Niacin ▪ Other nitrogenous compounds  A. Glucogenic amino acids Amino acids whose catabolism yields pyruvate or one of the intermediates of the citric acid cycle are termed glucogenic or glycogenic. These intermediates are substrates for gluconeogenesis and, therefore, can give rise to the net formation of glucose or glycogen in the liver and glycogen in the muscle.  B. Ketogenic amino acids Amino acids whose catabolism yields either acetoacetate or one of its precursor, (acetyl CoA or acetoacetyl CoA) are termed ketogenic. Acetoacetate is one of the ketone bodies, which also include 3-hydroxybutyrate and acetone. Leucine and lysine are the only exclusively ketogenic amino acids found in proteins. Their carbon skeletons are not substrates for gluconeogenesis and, therefore, cannot give rise to the net formation of glucose or glycogen in the liver, or glycogen in the muscle. C. Both glucogenic and ketogenic Isolucine, phenylalanine, tyrosine and tryptophan are glucogenic and ketogenic amino acids as part of its carbon skeleton can give glucose, the other can give ketone bodies  Protein Digestion Most of the nitrogen in the diet is consumed in the form of protein, typically amounting from 70 to 100g/day. Proteins are generally too large to be absorbed by the intestine. They must, therefore, be hydrolyzed to yield their constituent amino acids, which can be absorbed. Proteolytic enzymes responsible for degrading proteins are produced by three different organs: the stomach, the pancreas, and the small intestine Gastric Digestion Function of acidic pH Kills bacteria Denatures proteins Activation and Action of Pepsin Intestinal Digestion Pancreatic enzymes Intestinal enzymes 2. Pepsin – It is secreted by the chief cells in the form of inactive zymogen, pepsinogen. Pepsinogen is activated by HCl then by autocatalytic activation. HCl – Pepsinogen ⎯⎯⎯⎯⎯ → Pepsin – It is an endopeptidase with broad specificity, it is more specific to peptide bonds containing the carboxylic groups of aromatic amino acids (phenylalanine, tyrosine and tryptophan). Pepsin splits denatured proteins into large polypeptides.  In the small intestine Digestion in small intestine is due to the action of proteases present in both pancreatic and intestinal secretions. Pancreatic enzymes Pancreatic proteases are stored in proenzyme form in the pancreas. Pancreatic proteases have an optimum pH of 6-8. 1. Trypsin – Trypsin also activates other zymogens (chymotrypsinogen, proelastase and procarboxypeptidase). Trypsin is an endopeptidase ,acts specifically on peptide bonds connected to the carboxylic group of basic amino acids (arginine and lysine). Intestinal enteropeptidase Trypsinogen Trypsin  Dietary protein Pepsin Polypeptides and amino acids Stomach Trypsin Chymotrypsin Carboxypeptidase Elastase To liver Pancreas Oligopeptides and amino acids Aminopeptidases Small intestine Tripeptidase Dipeptidase Amino acids Digestion of dietary proteins by the proteolytic enzymes of the gastrointestinal tract.  Overall metabolism of nitrogen Nitrogen intake Nitrogen forms about 16% of proteins; normally one takes about 90 g of proteins, i.e., 14.5 g of nitrogen, daily. Nitrogen output After absorption, the amino acids of food proteins undergo many metabolic reactions. They are finally catabolized , chiefly by deamination, and their nitrogen is excreted through: urine, stools and other routes.  Nitrogen balance The nitrogen balance is the quantitative difference between the nitrogen intake and output. Since most of the nitrogen of the diet is protein nitrogen, and most of the nitrogenous excretory products are derived from protein catabolism, the balance between the two represents the balance between protein anabolism and catabolism. Three States may exist: 1- Nitrogen equilibrium. 2- Positive nitrogen balance. 3- Negative nitrogen balance.  Nitrogen equilibrium It exists when output equals intake. It occurs in the normal healthy adult on an adequate diet. 1- Positive nitrogen balance It exists when intake exceeds output. It occurs whenever new tissues are built, e.g., during growth, pregnancy, muscular training. 2. Negative nitrogen balance: as Inadequate protein intake – starvation, malnutrition, and gastrointestinal diseases. Loss of protein: as chronic hemorrhage, albuminuria, and during lactation, Increased protein catabolism – Also in diabetes mellitus, Cushing's syndrome, hyperthyroidism, and infectious fevers.  Transamination reactions The enzymes that catalyze this type of reaction are called aminotransferases and also called transaminases. They are present in the CYTOSOL and mitochondria of all tissues especially the liver. Transamination is the transfer of an amino group usually from an α-amino acid to an α-keto acid forming a new α-amino acid and a new α-keto acid. R.CH.COOH HOOC.CH2.CH2.C.COOH I II NH2 O transaminase α-Amino acid α-Ketoglutaric acid R.C.COOH HOOC.CH2.CH2.CH.COOH II I O NH2 α-Keto acid Glutamic acid 1. Alanine aminotransferase – The reaction is readily reversible. However, during amino acid catabolism, this enzyme functions in the direction of glutamate synthesis. CH3.CH.COOH HOOC.CH2.CH2.C.COOH I II NH2 O Alanine Alanine transaminase α-Ketoglutaric acid CH3.C.COOH HOOC.CH2.CH2.CH.COOH II I O NH2 Pyruvic acid Glutamic acid 2. Aspartate aminotransferase (AST) – aminotransferases funnel amino groups to form glutamate. During amino acid catabolism, AST transfers amino groups from glutamate to oxaloacetate, forming aspartate, which is used as a source of nitrogen in the urea cycle. HOOC.CH2.CH.COOH HOOC.CH2.CH2.C.COOH I II NH2 Aspartate O Aspartic acid transaminase α-Ketoglutaric acid HOOC.CH2.C.COOH HOOC.CH2.CH2.CH.COOH II I O NH2 Oxaloacetic acid Glutamic acid  Diagnostic value of plasma aminotransferases Aminotransferases are normally intracellular enzymes, with the low levels found in the plasma representing the release of cellular contents during normal cell turnover. The presence of elevated plasma levels of aminotransferases indicates damage to cells rich in these enzymes. Two aminotransferases AST and ALT are of particular diagnostic value when they are found in the plasma: 1. Liver disease: Plasma AST and ALT are elevated in nearly all liver diseases, but are particularly high in conditions that cause extensive cell necrosis, such as severe viral hepatitis, toxic injury, and prolonged circulatory collapse. 2. Nonhepatic disease: Aminotransferases may be elevated in nonhepatic disease, such as myocardial infarction and muscle disorders. Transport of ammonia to the liver Two mechanisms are available in humans for the transport of ammonia from the peripheral tissues to the liver for its ultimate conversion to urea: 1. The first, found in most tissues, uses glutamine synthetase to combine ammonia with glutamate to form glutamine,a nontoxic transport form of ammonia. The glutamine is transported in the blood to the liver where it is cleaved by glutaminase to produce glutamate and free ammonia.  2. The second transport mechanism, used primarily by muscle, involves transamination of pyruvate (the end-product of aerobic glyclosysis) to form alanine.Alanine is transported by the blood to the liver, where it is converted to pyruvate, again by transamination. In the liver, the pathway of gluconeogenesis can use the pyruvate to synthesize glucose, which can enter the blood and be used by muscle,a pathway called the glucose-alanine cycle.  From most tissues Urea H2O Glutamine Glutaminase Liver NH3 Glutamate dehydrogenase Glutamate α-ketoglutarate ALT Alanine Pyruvate Glucose From muscle Alanine cycle  Mechanism of ammonia toxicity Glucose The toxicity is thought to result, in part, from a Glycolysis shift in the equilibrium of the glutamate dehydrogenase reaction towards the direction of Pyruvate glutamate formation. This depletes α-ketoglutarate, an essential Oxaloacetate Acetyl CoA intermediate in the citric acid cycle, resulting in a decrease in cellular oxidation and ATP production. Citrate TCA The brain is particularly sensitive to hyperammonemia, because it depends on the NAD NH3 NADH Isocitrate NH3 citric acid cycle to maintain its high rate of energy Glutamine Glutamate α-ketoglutarate production. ADP ATP NAD NADH  Urea cycle This is the principal pathway of disposal of ammonia resulting from the deamination of amino acids. It allows the body to get rid of about 80-90% of the amino groups of amino acids in a neutral non-toxic form. Urea has no role in animal metabolism other than an end product that is excreted. Urea formation occurs only in the liver. Only the first 2 reactions occur in the mitochondria, all subsequent 3 reactions occur in the cytosol.  Mitochondrion 1 2 ATP + HCO3- + NH3 Carbamoyl phosphate + 2 ADP + Pi Pi Ornithine 2 Citrulline Citrulline Ornithine Urea cycle ATP 3 Aspartate Urea 5 AMP + PPi H2 O Arginino- Arginine succinate 4 Cytosol Fumarate Malat Oxaloacetate e  Hyperammonemi a The capacity of the hepatic urea cycle exceeds the normal rates of ammonia generation, and the levels of serum ammonia are normally low (5 to 50 pmol/L). However, when the liver function is compromised, due either to genetic defects of the urea cycle, or liver disease, blood levels can rise above 1000 mmol/L. Such hyperammonemia is a medical emergency, because ammonia has a direct neurotoxic effect on the CNS..