Proteins in the Diet and Nitrogen Metabolism (PDF)

Proteins in the Diet and Nitrogen Metabolism Dr. Önder Şirikçi Proteins in the Diet It was once beleived that “...the increase in dietary protein will lead to greater deposition of muscle & increased strength and vitality”. It is now known that an adequate protein intak...

Proteins in the Diet and Nitrogen Metabolism Dr. Önder Şirikçi Proteins in the Diet It was once beleived that “...the increase in dietary protein will lead to greater deposition of muscle & increased strength and vitality”. It is now known that an adequate protein intake is all that is necessary for health. In fact, excess intake is likely to be unhealthy. Protein Turnover Dietary protein (amino acids) Excess Loss - excretion Tissue protein Total amino acid pool Urea  Protein Turnover Dietary protein (amino acids) Total amino acid pool Tissue protein Excess Loss - excretion Urea  Protein Turnover All protein in the body are steadily synthesized and degraded (1-2 % of body protein/day) It is impossible to stop excretion, even when no protein is ingested in the diet When protein intake is high, excess amino acids are metabolized and excreted  Rate of Synthesis and Degradation Rapidly synthesized, rapidly degraded eg. plasma proteins, Rate of protein c albumin degradation may vary o Rapidly synthesized, have a finite n life, rapidly degraded eg. Hb between proteins c ...may vary according to e Long half-life, slow turnover eg. n structural proteins in brain or physiological demand (by t bone changing the rate of r degradation, thus a t partitioning metabolites i between pathways) o t ½ can vary from 30 s to n many days. time Rate of Synthesis and Degradation Protein turnover occurs in all forms of life Each day, 1-2 % of body protein is degraded in adults 75-80 % of liberated amino acids are reutilized for new protein synthesis. The N of the remainder is catabolized to urea, and the C skeleton to amphibolic intermediates Daily loss: 30-40 g/day or 5-7 g N  Replacing the Lost Amount (16 % (w/w) proteins is N. Proteins is the source of all dietary N. Total N x 6.25 = protein amount) Even when there is no protein intake: Total daily N loss = 54 mg N/kg body weight Total daily protein loss = 0.34 g protein/kg Total protein loss for 70 kg adult = 24 g/day  Replacing the Lost Amount Total protein loss for 70 kg adult = 24 g/day... ... is the minimum amount of protein that should be replaced for the lost amount, assuming a complete utilization If amino acids are utilized 70 % on average, the minimum daily intake should be 34 g for a 70 kg adult  No food which is pure protein. Foods are protein rich or protein poor  Quality of Protein Biological Value Nutritional value of a protein Egg 91 % assessed by measuring the ratio of Beef 67 % protein (N) content of the food taken Casein 56 % to the amount of N excreted (net protein utilization) Chemical Score IDEAL 100 % The ratio of essential amino acids in Egg 100 % a protein to the essential amino Beef 80 % Casein 80 % acids in the IDEAL protein  Essential Amino Acids Which one the amino acids below are Essential? Arginine** Threonine Histidine Tryptophan Isoleucine Methionine Leucine [Cysteine] * Lysine Phenylalanine Valine [Tyrosine] * * semi-essential; ** essential in young and growing Evaluation of Protein Nutrition Positive N balance N intake > N excretion (N is retained in the body; eg new protein synthesis) Negative N balance N intake < N excretion (N is lost from the body; eg protein breakdown) N equilibrium N intake = N excretion Effects of Dietary Intake High intake will induce protein catabolizing enzymes in the liver Amino acid catabolizing enzymes have a high Km (mM) and a low affinity for amino acids The tRNA synthetase enzymes have a low Km (M) and a high affinity for amino acids Oxidation of Amino Acids When do we see oxidation of amino acids ? During the normal turnover of body proteins When a protein rich diet is ingested, excess amino acids are catabolized and excreted When carbohydrates are unavailable or can not be utilized (starvation and diabetes) Nitrogen Is Important Only a few microorganisms can incorporate N2 to organic molecules NH3 is toxic for the nervous system Paths and Reactions in Amino Acid Catabolism Most amino acids are metabolized in the liver. Some of the NH3 is used, the rest is excreted. NH3 from other tissues is transported to the liver. Transamination Oxidative Deamination NH3 transport Urea synthesis  Transamination Reversible, both in synthesis and catabolism Specific for only one pair of -amino and -keto acid No net deamination or N loss from the amino acid pool Pyridoxine (Vit B6) Enzyme bound Schiff base intermediate  Glutamate Oxaloacetate Aminotransferase Channels N to glutamate Transaminases of Clinical Significance Asp + -KG AST, PLP OAA + Glu Ala + -KG ALT, PLP Pyruvate + Glu Specific for only one pair of -amino and -keto acid; -KG and Glutamate serve as amino group acceptor and donor in all amino transfer reactions. ALT is exclusively cytosolic AST is present both in cytoplasm and mitochondria as genetically distinct isoenzymes Glutamate Channels NH3 Flow In the hepatocyte cytosol, NH3 is transferred to KG. Glutamate then enters the mitochondria, where the amino group is removed to form NH4+. The glutamate functions as an amino group donor for biosynthetic pathways or excretion pathways. In muscle, excess NH3 is transferred to pyruvate to form alanine. Oxidative Deamination Hepatocyte mitochondria TCA Glucose Synthesis  Kidneys produce NH4+ coming from glutamine and not Liver mitochondria urea kidneys Excretion increases in metabolic acidosis, NH4+ forms salts with metabolic acids. Fates of NH3 NH3 in circulation is taken up by liver and channeled to; NH4 salts Urea Glutamate Glutamine Glucose Alanine Cycle Oxaloacetate Aspartate Urea Cycle  ARGININOSUCCINATE SYNTHETASE ORNITHINE TRANSCARBAMOYLASE ARGININOSUCCINATE LYASE ARGINASE CPS II; pyrimidine biosynthesis and is cytosolic  Aspartate – Argininosuccinate Shunt Fumarase & Malate dehydrogenase also occur in cytosol  Urea Cycle Takes place both in mitochondria and cytoplasm Mitochondrial and cytosolic enzymes are clustered to ensure efficiency. Citrulline is transported from the mitochondrion directly to the active site of argininosuccinate synthetase. Only urea is released into the cytosol.  Urea Cycle Activity Is Regulated N flux through the urea cycle varies with the composition of the diet. In protein rich, energy poor diets, the C skeletons of amino acids are used fuel, and much urea is produced from the excess NH3 groups. During starvation, urea production also increases  Short term regulation: Allosteric regulation of CPS I  Long term regulation: regulation of rate of synthesis of all 5 of the urea cycle enzymes Arginine any form of regulation where the regulatory molecule (an activator or inhibitor) binds to an enzyme someplace other than the active site How is urea cycle in short term regulated?  Energetics of Urea Cycle 2 NH4 + HCO3 + 3 ATP + H2O Urea + 2 ADP + 4Pi + AMP + 5 H2O NAD NADH  Synthesis of one molecule of urea requires 4 high energy phosphate bond cleavage.  However, the urea cycle also causes a net conversion of oxaloacetate to fumarate (via Asp) and the regeneration of oxaloacetate produces NADH, which can generate up to 2.5 ATP’s during mitochondrial respiration. Amino Acid Oxidation and the Catabolism of the Carbon Skeleton of Amino Acids Amino Acid Oxidation During the normal synthesis and degradation of cellular proteins When a diet rich in protein is ingested and the amino acids exceed the body’s need for new protein synthesis During starvation In Diabetes Mellitus Amino Acid Oxidation Amino acids lose their amino group to form the -keto acids, the “carbon skeletons” of amino acids. The -keto acids are oxidized to CO2 and H2O or, provide three or four carbon units that can be converted into glucose by gluconeogenesis Amino Acids and Their C Skeleton The Catabolism of the Carbon Skeleton of Amino Acids 20 catabolic pathways of amino acids converge to form 5 products, all of which enter the citric acid cycle. From here, the C skeletons are diverted to: Gluconeogenesis (pyruvate or TCA What happends to carbon of amino acids intermediate). in oxidation ? Ketogenesis (acetyl CoA). or are completely oxidized to H2O & CO2.  Glucogenic Glucogenic Ketogenic and ketogenic Nonessential Alanine Tyrosine Asparagine Aspartate Cysteine Glutamate Glutamine Glycine Proline Serine Essential Arginine Isoleucine Leucine Histidine Phenylalanine Lysine Methionine Tryptophan Threonine Valine  Enzyme Cofactors Amino group transfer PLP in transamination reactions Biotin transfers in the most oxidized state; CO2 Transfer of one carbon Tetrahydrofolate transfers units the intermediate states S-adenosylmethionine transfers in the most reduced state; CH3, Pyridoxine (Vit B6) Biotin Biotin transfers one-C units in its most oxidized state, CO2. Plays a key role in many carboxylation reactions. Pyruvate carboxylase Pyruvate + HCO3 + ATP Oxaloacetate + ADP + Pi The predominant form of CO2 at pH 7 is HCO3. Carboxyl groups are activated in a reaction that splits ATP and joins CO2 to enzyme bound biotin. The activated CO2 is then passed to an acceptor; pyruvate in this case. Role of Folic Acid in Amino Acid Metabolism Single C atoms can exist in a variety of oxidation states (methane, methanol, formaldehyde, formic acid, carbonic acid) It is possible to incorporate C units at each of these oxidation states (except methane) into organic compounds. These single C units can be transferred from carrier compounds (THF, SAM) to specific structures. THF transfers C units in intermediate oxidation states and sometimes as methyl groups  Synthesized in bacteria The one-C group transferred is is bonded to N-5, N-10, or both The most reduced form of one-C units on THF is N-5 methyl Intermediate N-5, N-10 methylene most oxidized forms are methenyl, formyl or formimino groups The major source of one-C units for THF is the C removed from Serine THF can carry a methyl, but its transfer potential is insufficient for most biosynthetic reactions Major source Preferred cofactor for biological methyl group transfers MS from some bacteria use N5 CH3-THF MS from mammals and other bacteria use N5 CH3-THF or methylcobalamine from vit B12 structure similar to folic acid (contains pterin) redox reactions instead of transfer reactions PKU Normal Plasma Amino Acid Chromatogram Phenylketonuria Tyrosinemia  Absent in liver Oxidized primarily in muscle, Branched chain -keto acids accumulate adipose, kidney and brain tissue in blood and are excreted in urine Maple Syrup Urine Disease Genetic Disorders of Amino Acid Catabolism Biosynthesis of Amino Acids During evolution, higher animals have lost the ability to synthesize amino acids whose formation requires protracted reaction sequences Most of the non-essential amino acids are synthesized from amphibolic intermediates by short pathways 3 of them (Cys, Tyr, Hyl) are formed from essential amino acids Number of Enzymes Required For Synthesis ESSENTIAL NON-ESSENTIAL Arg 7 Ala 1 His 6 Asp 1 Thr 6 Pro 3 Lys 8 Cys 2 Phe 10 Tyr 1