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TUMS Dr. Azin Nowrouzi Tehran University of Medical Sciences Many essential biomolecules contain N 2 Major Functions of Nitrogen Derived from Dietary Protein Heme Blood cell Choline PL Glycosamine Sugar Nucleotides DNA Protein synthesis Protein Biogenic amines Neurotransmitters Carnitine Heart Creatine phosphate « Energy » 3 The nitrogen cycle • No biomolecules are dedicated to the storage of N • excess N is excreted • N must be replenished by dietary protein 4 A. Ammonia Is Incorporated into Glutamate • Reductive amination of a-ketoglutarate by glutamate dehydrogenase occurs in plants, animals and microorganisms • In mammals & plants, located in mitochondria. 5 B. Glutamine Is a Nitrogen Carrier in Many Biosynthetic Reactions • A second important route in assimilation of ammonia is via glutamine synthetase • It is present in all organisms. • In humans it is most active in the liver. • Glutamine is transported from the liver to other tissues via the blood. 6 Assimilation of Ammonia Major Ammonium ion carrier 7 Glutamate synthetase • Glutamate synthatase is not present in humans. • It is found in Bacteria • It is used by blue-green algae and by Rhisobia. Transamination Reversible transfer of Ammonia between amino acids and a-ketoacids by aminotransferase • • Glutamate provides the amino group for the synthesis of many other amino acids through trasamination reactions Prosthetic group involved in amino transfer 9 Aspartate Aminotransferase Overall mechanism of transamination Alanine Aminotransferase 11 Central role of the aminotrasferases and glutamate dehydrogenase in nitrogen metabolism 1. Proteins are degraded to amino acids 2. Removal of nitrogen is first step in degradation 3. Ammonium is converted into urea 4. C-skeleton enters known pathways 5. Amino acids are made from intermediates of other pathways Amino Acid Synthesis • The ability of an organism to live and grow is dependent on protein synthesis • Therefore, a supply of all 20 aa is necessary. • Higher plants are able to synthesize all 20 aas. • Many microorganisms and higher animals make fewer • Humans make 10 of the 20 aas (these are called nonessential amino acids. • The remainder must be supplied in the diet, usually in the form of plant or animal proteins (these are called essential amino acids). Metabolic Classification of the Amino Acids • All 20 amino acids are essential for life, • They are necessary for protein synthesis – Essential or indispensable: 9 – Nonessential or dispensable: 11 • Review complete vs. incomplete • All natural, unprocessed animal and plant foods contain all twenty amino acids • A lack of any one of them leads to severe metabolic disruption and ultimate death. 14 What are the nonessential amino acids synthesized from? • Their synthesis depends on the availability of the appropriate carbon skeletons and a source of ammonia. • Glucose is ultimately the source of carbon skeletons for most nonessential aa. • Two essential aa, phenylalanine and methionine, are used to make tyrosine and cysteine, respectively. • Since ammonia is available in the fed state, amino acids become essential to our diet when we are not able to synthesize their carbon skeletons. α-keto acids required for synthesis of nonessential amino acids α-keto acid Pyruvate Oxaloacetate α-keto glutarate Pyruvate, 3-phosphoglycerate Amino acid Alanine Aspartate, Asparagine Glutamate, glutamine, Proline, Arginine Serine Biosynthesis of all amino acids in plants and microorganisms Biosynthesis of Amino Acids 18 Synthesis of Tyrosine Amino acids are precursors of some other biomolecules 20 Obtained in large amounts in diet as proteins Not like carbohydrate and fat most N is in proteins • Because no large stores of protein in the body AA’s are the source of N in the diet Amino Acids If not enough consumption Tissue breakdown occurs 21 Protein Quality Animal vs. Plant protein • Important in maintaining N balance • Proteins have different biological value (BV) • Major reasons why animal protein is called BV protein, whereas plant protein is of lower BV: – Animal protein is “complete” - contains all essential amino acids – Contains essential amino acids in larger amounts and in proper proportion for optimal utilization • Note: Soybean protein even though from a plant, is comparable to animal protein o In children leads to kwashiorker 22 28 grams 23 Note • The 56-g protein requirement for adult male can be met by 45 g of animal protein • Same requirement would necessitate 65 g plant protein • Combining plant products (legumes + grains) provides all essential amino acids • Mixture of 30% animal protein and 70 % plant protein similar to use of animal protein alone 24 Protein RDA varies in different stages of life cycle • • • • • • • 0-0.5 years: 1 g/lb 0.5-1 years: .71 g/lb 1-6 years: .56 g/lb 7-14 years: .45 g/lb 15-18 years: .41 g/lb 19+ years: .36 g/lb 1Ib = 0.4536 kg 25 26 Protein Turnover • Body proteins turn over; t1/2= min - wks • 400g of protein are synthesized per day and 400g are broken down – Secretory proteins such as digestive enzymes, polypeptide hormones, and antibodies, turn over rapidly – Structural proteins are much more metabolically stable. Chemical Signals for Turnover • ubiquitinatin – A small, heat stable protein (ubiquitin) reacts with other proteins to mark them for destruction • Oxidation of amino acid resides- Pro, Arg, Lys • Pest sequences- one or more regions rich in proline (P), gltamate (E),serine (S), and threonine (T) 28 Nitrogen Balance • No biomolecules are dedicated to the storage of N; • Excess N is excreted. • Definition: • Balances – Zero for healthy adults – Positive – – – – childhood growth pregnancy muscle building Healing – Negative for • Protein malnutrition • Essential aa malnutrition • “stress response” (fever, recovery from surgery, etc) 29 Protein Digestion • Gastric Digestion – Function of pH • Kills bacteria • Denatures proteins… – Activation and Action of Pepsin • Intestinal Digestion – Pancreatic enzymes – Intestinal enzymes 30 Denaturation of Proteins Protein Digestion in the Stomach at low pH Low pH N C Protein in Native Conformation Protein Denatured Pepsin N N N C C C N Oligopeptides C 31 Activation of the Gastric and Pancreatic Zymogens Trypsinogen Enteropeptidase Val-Asp-Asp-Asp-Asp-Lys Trypsin Chymotrypsinogen Proelastase Elastase Procarboxypeptidase Chymotrypsin Carboxypeptidase 32 Zymogens Activation slow, nonenzymatic + pepsinogen (zymogen) pepsin fast, enzymatic denatured proteins hydrolysis autocatalytic conversion of pepsinogen to pepsin large peptide fragments 33 34 Other details of Intestinal Enzymes Enzyme Occurrence pH optimum Major site of action Trypsin Intestine 7.5 to 8.5 Arginyl, lysyl bonds Chymotrypsin Intestine 7.5 to 8.5 Pepsin Stomach 1.5 to 2.5 Aromatic amino acyl bonds (Phe, Trp, Tyr) Wide range of specificity Carboxypeptidas e Intestine 7.5 to 8.5 C-terminal amino acid Aminopeptidase Intestinal mucosa N-terminal amino acid 35 Final conversion of peptides to free amino acids di- and tripeptides aa's aminopeptidase Brush Border di- and tripeptides peptidases amino acids 36 Amino Acid Absorption • Secondary active transport driven by Na+ gradient • Facilitated diffusion 37 Intestinal lumen Di- and Tripeptides Amino acid Na+ peptides peptidases Amino acid Na+ ATP ADP +Pi K+ Facilitated transporter Serosal side Brush border Na+ Active transporter Na+ K+ Transepithelial amino acid transport. Portal vein Amino acid 38 Gamma-Glutamyl Cycle • A metabolic cycle for transporting amino acids into cells. 39 Disorders of Amino Acid transportation or absorption Amino acid specificity Amino acids transported 1. Small neutral amino acids Alanine, serine, throenine 2. Large neutral and aromatic amino acids Isoleucine, leucine, valine, tyrosine, tryptophan, phenylalanine Arginine, lysine, omithine, cystine 3. Basic amino acids 4. Proline, glycine 5. Acidic amino acids Human disease Hartnup disease Cystinuria Glycinuria Glutamic and aspartic acids Uptake (transport) systems exist especially in intestine & kidney. Lack of specific transporter results in a disease state. 40 This can be partially overcome through uptake of peptides.