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Unformatted text preview: Biochemistry II (Bio 362) Lectures 1 & 2
Amino Acid Metabolism Dr. Karzai
Office: 244 CMM Learning is Not a Spectator Sport Your Full Participation is Required Key points:
Amino Acid biosynthesis: •-Intermediary metabolite precursors to non-essential AAs •-Incorporation of N into AAs •-Methyl Cycle, -THF, SAM •Amino acids as precursors Amino Acid Degradation: •-Flow of waste N from tissues to liver to Urea cycle •-Urea cycle •-Fate of carbon skeleton; Ketogenic vs. Glucogenic AAs Learning Objectives
• How is nitrogen “fixed” • Ammonium is assimilated into amino acids • Amino acids are made from precursors of major metabolic pathway • THF as carrier of several activated forms of one carbon species • SAM and the activated methyl cycle • Amino acids as precursors many essential Biomolecules • You are expected to know the structures of all 20 amino acids Essential & non-essential a.a. (for humans) Made by simple reactions Made by complex routes
(in bacteria and plants) Organisms vary greatly in their ability to synthesize the standard aa: most bacteria and plants synthesize all 20, mammals about half of them (remainder = “essential aa”, must come from food) Essential amino acids must be consumed in the diet. Mammalian cells lack enzymes to synthesize their carbon skeletons (α-keto acids). These include: Isoleucine, leucine, & valine Lysine Threonine Tryptophan Phenylalanine (Tyr can be made from Phe.) Methionine (Cys can be made from Met.) Histidine (Essential for infants.) Amino
Side chain Acid
R O C O H3N+ CH - Carboxylic acid Nitrogen is a Key Component of Amino Acids Nitrogen ﬁxation
Issue: N-N bond is very stable: bond energy = 225 kcal/mol
N2 + 3 H2 2 NH 3 : at 500°C, 300 ATM, Fe catalyst Industrial: Biological: N2 + 8 e- + 8 H+ + 16 ATP 2 NH3 + 16 ADP + 16 Pi + H2 at biological temps, 0.8 ATM Carried out by certain bacteria: - free-living soil bacteria - symbiotic bacteria in root nodules of legumes - cyanobacteria (blue-green algae) Symbiotic N2-fixing bacteria in nodules of legume roots
Root nodules Symbiotic bacteria inside root nodule cell nucleus NH4+ assimilation: glutamine & glutamate = entry points into metabolism Glutamate Dehydrogenase
This Reaction Proceeds in Two Steps Step One; Shiff base formation Step two; Shiff base is reduced by transfer of a hydride ion from NADP(H) adds a second ammonium ion to form Glutamine Glutamamine synthetase *ATP dependent reaction
Pay attention to how ATP is used. 6 Biosynthetic Families for aa
citric acid cycle Carbon skeletons come from intermediates in: •citric acid cycle: oxaloacetate, α-ketoglutarate. •glycolysis: PEP, pyruvate, 3-phosphoglycerate. •pentose-P pathway: ribose-5-P, erythrose-4-P.
Glycolysis Pentose-P pathway Glycolysis + pentose-P pathway Citric acid cycle Glycolysis Note: - some aa are precursors of others (red boxes) - essential aa in bold Biosynthesis of Asparagine from Aspartate *ATP dependent reaction Ser, Cystein & Gly are formed from 3-phosphoglycerate Cysteine N N l H 2C CH2 l N- Biosynthesis of Cys from Ser Glutamate & glutamine as N-donors
Glutamate: The α-amino group is the source of the α-amino group in most other amino acids - Amino transfer reactions are catalyzed by aminotransferases that all require a cofactor (pyridoxal phosphate; B6) Glutamine donates its side-chain N (= amide N) in the biosynthesis of a wide range of compounds - reactions are catalyzed by glutamine amidotransferases Classes of reactions involved in aa & nucleotide synthesis
1. Transamination reactions (w. glutamate) 2. Transfer of amino groups derived from amide N of glutamine 3. Transfer of 1 carbon groups using tetrahydrofolate as cofactor Transaminase enzymes (aminotransferases) catalyze the reversible transfer of an amino group between two α-keto acids. COO− COO− CH2 HC NH3+ CH2 CH2 COO− CH2 O C O COO− CH2 CH2 + C + HC N H 3+ COO− COO− COO− COO− aspartate -ketoglutarate oxaloacetate glutamate Aminotransferase (Transaminase)
Example of a Transaminase reaction: Aspartate donates its amino group, becoming the α-keto acid oxaloacetate. α-Ketoglutarate accepts the amino group, becoming the amino acid glutamate. In another example, alanine becomes pyruvate as the amino group is transferred to α-ketoglutarate. Transaminases equilibrate amino groups among available α-keto acids. This permits synthesis of non-essential amino acids, using amino groups from other amino acids & carbon skeletons synthesized in a cell. Thus a balance of different amino acids is maintained, as proteins of varied amino acid contents are synthesized. Although the amino N of one amino acid can be used to synthesize another amino acid, N must be obtained in the diet as amino acids (proteins). The prosthetic group of Transaminase is pyridoxal phosphate (PLP), a derivative of vitamin B6. R H C NH2 COO− Enz (CH2)4 Amino a cid
−O O O− P O HC H2 C + N H N+ H O− CH3 Enzyme (Lys)-PLP Schiff base In the resting state, the aldehyde group of pyridoxal phosphate is in a Schiff base linkage to the ε-amino group of an enzyme lysine residue. The α-amino group of a substrate amino acid displaces the enzyme lysine, to form a Schiff base linkage to PLP. Enz−Lys−NH2 R HC H C N+ COO− H O− −O O O− P O H2 C + N H CH3 Amino acid-PLP Shiff base (aldimine) The active site lysine extracts H+, promoting tautomerization, followed by re-protonation & hydrolysis. What was an amino acid leaves as an α-keto acid. The amino group remains on what is now pyridoxamine phosphate (PMP). A different α-keto acid reacts with PMP and the process reverses, to complete the reaction. Several other enzymes that catalyze metabolism or synthesis of amino acids also utilize PLP as prosthetic group, and have mechanisms involving a Schiff base linkage of the amino group to PLP. In addition to equilibrating amino groups among available α-keto acids, transaminases funnel amino groups from excess dietary amino acids to those amino acids (e.g., glutamate) that can be deaminated. Carbon skeletons of deaminated amino acids can be catabolized for energy, or used to synthesize glucose or fatty acids for energy storage. Only a few amino acids are deaminated directly. Glutamate Dehydrogenase catalyzes a major reaction that affects net removal of N from the amino acid pool.
It is one of the few enzymes that can use NAD+ or NADP+ as e− acceptor. Oxidation at the α-carbon is followed by hydrolysis, releasing NH4+. Tetrahydrofolate: carries activated forms of 1-carbon groups N N H
5 N CH2 l 10 NH N CH2 l N- N N CH2 l N- N + N CH2 l N- N N CH2 l N- N N CH2 l N- N N l
H 3C l
H 2C ll
O=CH l HC l ll
HN CH2 l N- O=CH N5, N10 methylene THF
USED IN GLYCINE BIOSYNTHESIS & IN CONVERSION OF URIDINE TO THYMIDINE N10 formyl THF
USED 2 X IN PURINE BIOSYNTHESIS S-adenosylmethionine “SAM” = preferred methyl donor
Involved in: - ethanolamine −> choline methylation - “capping” of messenger RNA Presence of positively-charged sulfonium ion ( powerful alkylating agent: S+ ) makes SAM a the methyl group is readily attacked by nucleophiles [much more reactive as methyl donor than N5-methyl tetrahydrofolate] Formation of S-Adenosylmethionine Methyl Donor Reaction The SAM Methyl Cycle N5 THF Amino Acids as Precursors of Important Biomolecules ANABOLIC PATHWAYS THAT UTILIZE AMINO ACIDS
A. Biosynthesis of creatine
arginine + glycine guanidino acetic acid S-adenosyl-L-methionine creatine B. Phenylalanine and tyrosine are precursors of the catecholamines, dopamine, norepinephrine, and epinephrine. Clinical ramifications involving dopamine: Dopamine has a role in the pathogenic mechanisms that cause Parkinson’s disease. Parkinson’s disease is characterized by a loss of dopaminergic neurons that results in diminished levels of dopamine in the striatum. Biosynthesis of the catecholamines phenylalanine tyrosin
Tyrosine 3-monooxygenase* dihydroxyphenylalanine (DOPA) dopamine norepinephrine
*Rate-determining step C. Tryptophan is the precursor of 5-hydroxy-tryptamine (serotonin).
Clinical ramifications The broad actions of the neurotransmitter 5-hydroxytryptamine involve effects on emotion, mood, and reward. Suicide and depression have been associated with reduced serotonergic transmission. D. Arginine is the precursor of the second messenger nitric oxide (NO). This is a second messenger that appears to have distinctly different functions in different cell types. For example, the nitric oxide synthesized by platelets is seen to inhibit platelet aggregation and adherence. In this way it contributes to the antithrombogenic properties of the endothelium. In the kidney (in experimental animals), a diminution in nitric oxide was associated with glucocorticoidinduced hypertension. Such studies were carried out because it had been suggested that decreased nitric oxide contributes to the impaired endothelium-dependent vasodilatation seen in essential hypertension. How does it work?
NO diffuses across the muscle cell membrane and binds to guanylyl cyclase. Guanylyl cyclase catalyzes the synthesis of cyclic GMP from GTP. cGMP then activates a cGMP dependent protein kinase which in turn stimulates the uptake of calcium by the endoplasmic reticulum of the muscle cell. The reduced levels of cytoplasmic calcium cause the muscle cell to relax. As a consequence of muscle cell relaxation, vasodilation occurs. As is true of any signaling pathway, there must be a way to terminate the action of the signal. cGMP is converted into GMP by a specific phosphodiesterase (PDE.) There are 10 families of PDEs: PDE1-10. The major PDE found in vascular smooth muscle is PDE5. Viagra (sildenafil) is a specific inhibitor of PDE5. By blocking the breakdown of cGMP, Viagra acts to prolong the effects of cGMP E. Methionine has multiple anabolic fates. 1. As a precursor of the polyamines, spermidine and spermine. While it is generally accepted that the polyamines, spermidine and spermine, are essential for cell proliferation in mammalian cells, the critical reaction(s) have not been identified. Inhibiting ornithine decarboxylase (ODC) (the ratedetermining enzyme) has been shown to inhibit cell growth. 2. As a precursor of cysteine Cysteine is synthesized in humans using the carbons of serine and the sulfur of methionine. METHIONINE SERINE + Homocysteine CYSTEINE 3. As a precursor of S-adenosyl-L-methionine (SAM) The methyl donor S-adenosyl-L-methionine is derived from methionine. METHIONINE + ATP S-ADENOSYL-L-METHIONINE + Pi + PPi 4. As a necessary participant in the synthesis of deoxyribothymidylic acid and, hence, DNA The conversion of deoxyribouridylic acid monophosphate (dUMP) into the corresponding thymidylic acid derivative (dTMP) depends upon converting 5methyltetrahydrofolate into the unligated tetrahydrofolate. This is accomplished by the enzyme methionine synthase. In summary, methionine provides carbon and nitrogen atoms that become a part of spermidine and spermine, the sulfur atom that is required for the biosynthesis of cysteine, and the “active” methyl group of the methyl donor, S-adenosyl-L-methionine. H3C ─S NH O CH2─CH2─CH──C─OH ...
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This note was uploaded on 03/16/2010 for the course BIO 362 taught by Professor Walikarzai during the Spring '10 term at SUNY Stony Brook.
- Spring '10