Unformatted text preview: Lectures 36 and 37 Nitrogen Metabolism Bioc 460 Spring 2010 Dr. Lisa Rezende How is nitrogen fixed and assimilated from the air? How do organisms maintain nitrogen balance? How are non-essen8al amino acids synthesized? Image credit: EPA Learning ObjecDves Explain, on a biochemical level, how bacteria fix nitrogen from the atmosphere. Describe how proteins are digested in animal cells. Describe the transaminase, and glutamate dehydrogenase reac8ons and discuss their roles in the removal of nitrogen waste in the body. Describe the urea cycle by wri8ng the balanced chemical reac8on, naming the regulated step, and explaining how it is integrated with the citrate cycle and gluconeogenesis. Describe the phenylalanine hydroxylase reac8on and explain its rela8onship to phenylketonuria. Explain how amino acid degrada8on is related to gluconeogenesis and ketosis. Recognize the difference between essen8al amino acids and nonessen8al amino acids and explain why the list is different for different organisms. Discuss the biochemical basis of diseases, drugs, and poisons that target amino acid synthesis pathways in a variety of organisms. Describe the basic steps in heme biosynthesis and degrada8on, and discuss diseases associated with defects in heme metabolism. Reading: Chapters 23 and 24 Problems: Chapter 23- 10, 11, 12, 13, 18 plus problem set Amino Acid Metabolism Nitrogen Fixa8on N2 + 8 H+ 8 e- + 16 ATP + 16 H2O ----> 2 NH3 + H2 + 16 ADP + 16 Pi Urea forma8on NH4+ + HCO3- + aspartate + 3 ATP ---> urea + fumarate + 2 ADP + 2 Pi + AMP + PPi Courtesy of R.L. Miesfeld Atmospheric Nitrogen(N2) is Fixed by Bacteria Nitrogen fixa8on produces ammonium (NH4), nitrates (NO3-), and Nitrites (NO2-) That are assimilated into nitrogenous biomolecules (amino acids and nucleo8des). Nitrogen-fixing bacteria o]en from symbio8c rela8onships with plants. Photo credit: h^p://academic.reed.edu/biology/Nitrogen/index.html Nitrogen fixa8on requires ATP and electrons! N2 + 8 H+ 8 e- + 16 ATP + 16 H2O ----> 2 NH3 + H2 + 16 ADP + 16 Pi Nitrogen fixaDon in bacteria Biological nitrogen fixa8on by bacteria requires the ac8vity of nitrogenase, a large protein complex consis8ng of two func8onal components. One component is called dinitrogenase reductase (Fe-protein) which consists of two iden8cal subunits that each contain a binding site for ATP, and a single 4Fe-4S redox center liganded to cysteine residues in the two subunits. Courtesy of R.L. Miesfeld Nitorgenase Complex is SensiDve to O2 Leghemoglobin, a homolog of hemoglobin, present in the root nodules of leguminous plants binds O2 What other enzyme have we discussed that must be kept away from O2? Nitrogenase reacDon is a series of reducDons N2 + 8 H+ 8 e- + 16 ATP + 16 H2O ----> 2 NH3 + H2 + 16 ADP + 16 Pi Courtesy of R.L. Miesfeld Nitrogen assimilaDon in plants Once atmospheric nitrogen has been fixed into ammonium, nitrogen assimila8on proceeds in one of two ways. First, if NH4+ levels in the soil are high, plants can use the glutamate dehydrogenase reac8on to directly incorporate NH4+ into the amino acid glutamate using - ketoglutarate as the carbon skeleton. Note that most o+en, this reac1on releases NH4+ from glutamate in other contexts. Courtesy of R.L. Miesfeld Where does -ketoglutarate come from? Nitrogen assimilaDon in plants A second, and more common way that plants and bacteria incorporate NH4+ into metabolites, is through a two reac8on mechanism that func8ons when NH4+ concentra8ons are low. In this mechanism, the enzyme glutamine synthetase uses ATP in a coupled reac8on to form glutamine from glutamate using NH4+. Courtesy of R.L. Miesfeld Nitrogen assimilaDon in plants Next, the glutamine is combined with -ketoglutarate in a reac8on catalyzed by the enzyme glutamate synthase to form two molecules of glutamate (glutamine contains two nitrogens). Courtesy of R.L. Miesfeld Importantly, the newly acquired nitrogen in glutamate and glutamine is used to synthesize a variety of other amino acids through aminotransferase enzymes such as aspartate aminotransferase. Courtesy of R.L. Miesfeld Dietary protein provides nitrogen for humans A normal healthy adult needs about 400 grams of protein per day to maintain nitrogen balance. In contrast, young children and pregnant women have a posiDve nitrogen balance because they accumulate nitrogen in the form of new protein which is needed to support 8ssue growth. NegaDve nitrogen balance is a sign of disease or starva8on and occurs in individuals with elevated rates of protein breakdown (loss of muscle 8ssue) or an inability to obtain sufficient amounts of amino acids in their diet. Courtesy of R.L. Miesfeld Protein digesDon in humans takes place in the stomach and the small intes8ne where proteases cleave the pepDde bond to yield amino acids and small oligopep8des. (Recall proteoly8c ac8va8on from the protein regula8on lecture) Courtesy of R.L. Miesfeld Amino acids are stripped of their carbon Courtesy of R.L. Miesfeld The Urea Cycle Courtesy of R.L. Miesfeld In the liver, many amino acids are converted to glutamate, which is then deaminated Toxic, must be removed! NH4+ is converted to urea via the urea cycle NH4+ produced by the glutamate dehydrogenase catalyzed reac8on is used to make the urea cycle precursor carbamoyl phosphate. The Urea Cycle Occurs primarily in the liver Regulated step is catalyzed by carbomoyl phosphate synthetase which is allosterically ac8vated by acetylglutamate acetyl CoA + glutamate -> N-acetylglutamate What is the metabolic logic of this regulatory step? Fumarate (returns to TCA cycle) Arginine Argininosuccinate AMP+PPi O UREA H2N-C- NH2 2ATP + HCO3- + NH4+ Ornithine Citrulline Ornithine Carbamoyl phosphate synthetase 2ADP + Pi -Aspartate -OOC-CH- Carbamoyl phosphate Ornithine transcarbamoylase Citrulline Pi MITOCHONDRIA CH2COO- NH + 3 ATP CYTOPLASM argininosuccinate synthetase Modified from a figure courtesy of Dr. M. Tischler argininosuccinase arginase The aspartate-argininosuccinate shunt links the urea cycle and glucoenogenesis DegradaDon of glucogenic and ketogenic amino acids The carbon backbones of eleven of the twenty standard amino acids can be converted into pyruvate or acetyl-CoA, which can then be used for energy conversion by the citrate cycle and oxida8ve phosphoryla8on reac8ons. The other nine amino acids are converted to the citrate cycle intermediates ketoglutarate, fumarate, succinyl-CoA, and oxaloacetate, which can be used for glucose synthesis by conversion of oxaloacetate to phosphoenolypyruvate. Under normal condi8ons, amino acid degrada8on accounts for ~10-15% of the metabolic fuel for animals, more so for animals with high protein diets or during starva8on when muscle protein is degraded. Courtesy of R.L. Miesfeld The carbon skeletons of amino acids Enter the citrate cycle at various intermediates Glucogenic (pink) and ketogenic (yellow) amino acids Note that several can be both glucogenic and ketogenic Overview of Amino Acid Biosynthesis Arginine is listed as an essen8al amino acid because humans require arginine in their diet to support rapid growth during childhood and pregnancy, even though it is made by urea cycle. Tyrosine is also highlighted because this condi6onal nonessenDal amino acid is made in humans from the essen8al amino acid phenylalanine. Courtesy of R.L. Miesfeld In general, the structures of the essenDal amino acids are more complex than the nonessenDal amino acids which is reflected in the number of enzyma8c reac8ons required for synthesis. Adapted from R.L. Miesfeld Amino acid biosynthesis is regulated by Feedback inhibi8on Herbicides take advantages of pathways that exist in plants but not animals One of the most widely used herbicides is glyphosate, the ac8ve ingredient in Roundup. Glyphosate is a compe88ve inhibitor of the enzyme EPSP synthase which is required to convert shikimate 3-phosphate to EPSP. Since animals do not have the shikimate pathway enzymes, Roundup is an animal safe herbicide. Do you think glyphosate works faster in the summer or winter? Explain. Courtesy of R.L. Miesfeld Roundup Ready Crops are Glyphosate-Resistant Monsanto developed glyphosate-resistant crop plants so that farmers could spray their transgenic crops with Roundup and kill weeds that reduce crop yields without harming the crop plants. The first glyphosate-resistant crop plant developed was a strain of soybeans marketed as Roundup Ready soybeans. Courtesy of R.L. Miesfeld Amino Acids as Metabolic Precursors Because of the nitrogen content of amino acids (the -amino group), they are also used as metabolic precursors for numerous biomolecules, including: Heme groups (hemoglobin and cytochromes), NucleoDde bases (purines and pyrimidines) Signaling molecules (neurotransmi^ers, hormones, nitric oxide). Courtesy of R.L. Miesfeld Amino Acids as Metabolic Precursors Tyrosine is the precursor to several important molecules in metabolic signaling and neurotransmission, including epinephrine and dopamine. Courtesy of R.L. Miesfeld Tyrosine is also the precursor to pigment molecules called melanins that are produced from dopaquinone. Courtesy of R.L. Miesfeld A gene8c defect in the gene encoding phenylalanine hydroxylase is responsible for the metabolic disease phenylketonuria (PKU). Courtesy of R.L. Miesfeld The clinical symptoms of PKU are caused by the accumula8on of phenylalanine in the blood that is 30-50 Dmes higher than normal. Phenylketonuriacs have to be careful to avoid processed foods and beverages containing the food addi8ve aspartame (aspartyl- phenylalanine methyl ester). Courtesy of R.L. Miesfeld Type 1 albinism is an autosomal recessive geneDc mutaDon in the tyrosinase gene Interes8ngly, individuals with phenylketonuria can have light skin and hair at birth because of low levels of tyrosine. However, phenylketonuriacs are not albinos because they obtain sufficient amounts of tyrosine in their diets to support melanin biosynthesis. Why is PKU treatable, but albinism is not, even though both are the result of geneDc mutaDons in enzymes? Courtesy of R.L. Miesfeld Heme Biosynthesis 8 Tracing the carbon skeletons That contribute to heme Congenital porphyrias effect heme biosynthesis and can be the result of dominant mutaDons How might a mutant protein cause a dominant (gain of funcDon) phenotype? Courtesy of R.L. Miesfeld Heme Degreda8on ...
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