Exam 4 Review PDF

Review for Exam 4 Exam 4: Lectures 22 to 26 and JCs 9 and 10 TWO CONVENTIONS FOR NAMING FATTY ACIDS (a) Standard convention: assigns the number 1 to the carboxyl carbon (C-1), and α to the carbon next to it. The position of any double bond(s) is indicated by Δ followed by a superscript number indica...

Review for Exam 4 Exam 4: Lectures 22 to 26 and JCs 9 and 10 TWO CONVENTIONS FOR NAMING FATTY ACIDS (a) Standard convention: assigns the number 1 to the carboxyl carbon (C-1), and α to the carbon next to it. The position of any double bond(s) is indicated by Δ followed by a superscript number indicating the lower-numbered carbon in the double bond. Examples: Palmitic acid, a C16 saturated fatty acid - 16:0 Palmitoleic acid, a C16 mono-unsaturated - 16:1(D9) Oleic acid, a C18 mono-unsaturated - 18:1(D9) (a) Alternative convention: For polyunsaturated fatty acids (PUFAs):, this convention numbers the carbons in the opposite direction, assigning the number 1 to the methyl carbon at the other end of the chain; this carbon is also designated ω (omega; the last letter in the Greek alphabet). The positions of the double bonds are indicated relative to the ω carbon. ω-3 fatty acids are essential nutrients. Humans need them but cannot synthesize them, including ALA, DHA, and EPA (although DHA and EPA can be synthesized from ALA) ALA Alpha Linolenic Acid; 18:3(D9,12,15) (ALA) (soybeans, flaxseeds, walnuts), Eicosa Pentanoic Acid; 20:5(D5,8,11,14,17) (EPA), (fish oil), Docosa Hexanoic Acid; 22:6(D4,7,10,13,16,19) (DHA) (fish oil), are essential Omega-3 fatty acid (although EPA and DHA can be made from ALA). CLASSIFICATION OF LIPIDS Based on the structure and function Lipids that do not contain fatty acids: cholesterol, terpenes, … Lipids that contain fatty acids (complex lipids) – can be further separated into: – storage lipids and membrane lipids Structural lipids All the lipid types shown here have either glycerol or sphingosine as the backbone (pink screen), to which are attached one or more long-chain alkyl groups (yellow) and a polar head group (blue). - In triacylglycerols, glycerophospholipids, galactolipids, and sulfolipids, the alkyl groups are fatty acids in ester linkage. - Sphingolipids contain a single fatty acid, in amide linkage to the sphingosine backbone. The membrane lipids of archaea are variable; that shown here has two very long, branched alkyl chains, each end in ether linkage with a glycerol moiety. In phospholipids the polar head group is joined through a phosphodiester, whereas glycolipids have a direct glycosidic linkage between the head-group sugar and the backbone glycerol. 2/ STRUCTURAL LIPIDS (phospholipids or membrane lipids) Glycerol based = Glycerophospholipids Sphingosine based = Sphingolipids GLYCEROPHOSPHOLIPIDS ARE STRUCTURAL LIPIDS IN MEMBRANES (POLAR) Contain polar head groups and nonpolar tails (usually attached fatty acids) Diversification can come from: using a different backbone changing the fatty acids modifying the head groups Non-polar acyl chains Polar headgroup Backbone The properties of head groups determine the surface properties of membranes Different organisms have different membrane lipid head group compositions Different tissues have different membrane lipid head group compositions EXAMPLES OF GLYCEROPHOSPHOLIPIDS Glycerophospholipids are named as derivatives of the parent compound, phosphatidic acid, according to the polar alcohol in the headgroup Glycerophospholipids: The common glycerophospholipids are diacylglycerols linked to head-group alcohols through a phosphodiester bond. Phosphatidic acid, a phosphomonoester, is the parent compound. Each derivative is named for the head-group alcohol (X), with the prefix "phosphatidyl-." In cardiolipin, two phosphatidic acids share a single glycerol (R1 and R2 are fatty acyl groups). SPHINGOLIPIDS The backbone of sphingolipids is NOT glycerol The backbone of sphingolipids is sphingosine, a long-chain amino alcohol: A fatty acid, usually saturated or monounsaturated, with 16, 18, 22, or 24 carbon atoms, is joined to sphingosine via an amide linkage rather than an ester linkage, as usually seen in lipids. A polar head group is connected to sphingosine by – A phosphodiester linkage (Ex: sphingomyelin) – A glycosidic linkage (one, or more, sugar directly attached to the OH at C-1 (Ex: glucosyl-ceramide). The sugarcontaining glycosphingolipids are found largely in the outer face of plasma membranes SPHINGOLIPIDS: DERIVATIVES OF SPHINGOSINE The first three carbons at the polar end of sphingosine are analogous to the three carbons of glycerol in glycerophospholipids. The amino group at C-2 bears a fatty acid in amide linkage. The fatty acid is usually saturated or monounsaturated, with 16, 18, 22, or 24 carbon atoms. Ceramide is the parent compound for this group. Other sphingolipids differ in the polar head group (X) attached at C-1. Gangliosides have very complex oligosaccharide head groups. SPHINGOMYELIN – A PHOSPHOLIPID WITH A SPHINGOSINE BACKBONE Ceramide (sphingosine + amide-linked fatty acid) + phosphocholine attached to the alcohol Sphingomyelin is abundant in myelin sheath that surrounds some nerve cells in animals SPHINGOMYELIN IS STRUCTURALLY SIMILAR TO PHOSPHATIDYLCHOLINE choline sphingosine Amide-linked fatty acid 1 2 3 PC Sphingomyelin (a sphingolipid) and phosphatidylcholine, PC, (a glycerophospholipid) have similar dimensions and physical properties, but presumably play different roles in membranes. PHOSPHOLIPIDS AND SPHINGOLIPIDS ARE DEGRADED IN LYSOSOMES Specifity of Phospholipases Phospholipases A1 and A2 (PLA1, PLA2) hydrolyze the ester bonds of intact glycerophospholipids at C-1 and C-2 of glycerol, respectively. When one of the fatty acids has been removed by a type A phospholipase, the second fatty acid is removed by a lysophospholipase (not shown). Phospholipases C and D (PLC, PLD) each split one of the phosphodiester bonds in the head group. Some phospholipases act on only one type of glycerophospholipid, such as phosphatidylinositol 4,5-bisphosphate (shown here) or phosphatidylcholine; others are less specific § PLC releases signaling molecules (PIP2 -> IP3 + DAG) § PLA2 releases a fatty acid from the C-2 position of glycerol; the C-2 fatty acid that is released is often arachidonic acid [used for synthesis of eicosanoids] JC10: LYSOSOMAL STORAGE DISEASES DEFECTS IN THE TURNOVER OF MEMBRANE LIPIDS LEAD TO A NUMBER OF DISEASES Pathways for the breakdown of GM1, globoside, and sphingomyelin to ceramide. A defect in the enzyme hydrolyzing a particular step is indicated by V; the disease that results from accumulation of the partial breakdown product is noted. § Sterols: STEROLS AND CHOLESTEROL § Steroid nucleus - 4 fused rings, almost planar, relatively rigid § Made from isoprene units § Amphipathic (polar: OH at C-3, non-polar hydrocarbon side-chain at C-17) § Hydroxyl group (polar head) at C-3 in the A-ring § Various nonpolar side chains at C-17 § Synthesized from acetyl CoA via 5-carbon isoprene units § Cholesterol: major sterol in animals §Structural lipid; component of membranes Acetyl CoA H2C H3C Cholesterol: The C-3 hydroxyl group (pink in both representations) is the polar head group. H C CH2 Isoprene units For storage and transport of the sterol, this hydroxyl group condenses with a fatty acid to form a sterol ester. PHYSIOLOGICAL ROLE OF STEROLS Cholesterol and related sterols are present in the membranes of most eukaryotic cells – Modulate fluidity and permeability – Thicken the plasma membrane – Most bacteria lack sterols Mammals obtain cholesterol from food or synthesize it de novo in the liver Cholesterol, bound to proteins, is transported to tissues via blood vessels – Cholesterol in low-density lipoproteins (LDL) tends to deposit and clog arteries Many hormones are derivatives of sterols BILE ACIDS ARE POLAR DERIVATIVES OF CHOLESTEROL §Produced in the liver and stored in the gallbaldder § Act as detergents in the intestine, emulsifying dietary fats and making them more accessible to lipases Cholic acid (made in the liver) One of cholic acid derivatives Emulsification of fat by Bile From Understanding Nutrition, 14th edition BIOLOGICALLY ACTIVE LIPIDS Present in much smaller amounts than storage or structural lipids Play vital roles as signaling molecules between nearby cells (paracrine hormones) Lipid soluble vitamins (A, D, E, and K) EICOSANOIDS (SIGNALING LIPIDS) ARE DERIVATIVES OF ARACHIDONIC ACID § Eicosanoids: Paracrine hormones - act near the site of hormone synthesis § Carry messages to nearby cells § mediate inflammation, vasoconstriction and platelet aggregation § Enzymatic oxidation of arachidonic acid yields 3 classes of derivatives: §Prostaglandins, thromboxanes, leukotrienes § Non-steroidal anti-inflammatory drugs (NSAIDs; aspirin, ibuprofen) block cyclooxygenase (COX)-mediated conversion of arachidonate to prostaglandins and thromboxanes Nonsteroidal antiinflammatory drugs (NSAIDs) such as aspirin and ibuprofen block the formation of prostaglandins and thromboxanes from arachidonate by inhibiting the enzyme cyclooxygenase (prostaglandin H2 synthase). Arachidonic acid (20:4(D5,8,11,14)) and its derivatives - eicosanoids VITAMIN D (CHOLECALCIFEROL) REGULATES CALCIUM UPTAKE PLAYS A ROLE IN IMMUNITY Fat soluble hormone precursor modulates the innate and adaptive immune response Active hormone Vitamin D3 production and metabolism. Cholecalciferol (vitamin D3) is produced in the skin by UV irradiation of 7-dehydrocholesterol, which breaks the bond shaded light red. In the liver, a hydroxyl group is added at C-25; in the kidney, a second hydroxylation at C-1 produces the active hormone, 1α,25-dihydroxyvitamin D3. This hormone regulates the metabolism of Ca2+ in kidney, intestine, and bone. Deficiency leads to poor bone mineralization: rickets in children and oteomalacia in adults. Overdose à Hypercalcemia à anorexia, nausea, polyuria, weakness, DERIVATIVES OF VITAMIN A (RETINOL) PLAY A ROLE IN VISUAL TRANSDUCTION AND IN GENE EXPRESSION Fat soluble Precursor for other hormones involved in signaling Vitamin A1 and its precursor and derivatives β-Carotene is the precursor of vitamin A1. Isoprene structural units are set off by dashed red lines). Cleavage of β-carotene yields two molecules of vitamin A1 (retinol) (b). Oxidation at C-15 converts retinol to the aldehyde, retinal (c), Further oxidation produces retinoic acid (d), a hormone that regulates gene expression. Retinal combines with the protein opsin to form rhodopsin, a visual pigment widespread in nature. Deficiency leads to blindness hyperkeratosis and anemia... Overdose à liver dysfunction and osteoporosis.. In the dark, retinal of rhodopsin is in the 11-cis form (c). When a rhodopsin molecule is excited by visible light, the 11-cis-retinal undergoes a series of photochemical reactions that convert it to all-trans-retinal (e), forcing a change in the shape of the entire rhodopsin molecule. This transformation in the rod cell of the vertebrate retina sends an electrical signal to the brain that is the basis of visual transduction. VITAMIN E (a-TOCOPHEROL), VITAMIN K AND OTHER LIPID QUINONES ARE ANTIOXIDANTS Fat soluble Diverse roles Vitamin E removes free radicals. Protection against nonenzymatic oxidation of macromolecules Vitamin K1 (phylloquinone) is involed in carboxyglutamate formation involved in the activation of blood-clotting factors. Isoprenoids derivatives: Units derived from isoprene are set off by dashed red lines. In most mammalian tissues, Ubiquinone (also called coenzyme Q) has 10 isoprene units. Dolichols of animals have 17 to 21 isoprene units (85 to 105 carbon atoms), DIETARY TRIACYLGLYCEROLS ARE ABSORBED IN THE SMALL INTESTINE: CYCLES OF RELEASE AND ESTERIFICATION Processing of dietary lipids in vertebrates. Digestion and absorption of dietary lipids occur in the small intestine, and the fatty acids released from triacylglycerols are packaged and delivered to muscle and adipose tissues. DIETARY LIPIDS ARE TRANSPORTED IN THE BLOOD AS CHYLOMICRONS Molecular structure of a chylomicron The surface is a layer of phospholipids, with head groups facing the aqueous phase. Triacylglycerols sequestered in the interior (yellow) make up more than 80% of the mass. Several apolipoproteins that protrude from the surface (B-48, C-III, C-II) act as signals in the uptake and metabolism of chylomicron contents. The diameter of chylomicrons ranges from about 100 to 500 nm. IN THE LIVER, LIPIDS ARE PACKAGED IN VLDL, LDL AND HDL From Understanding Nutrition, 14th edition Lipid transport via lipoproteins: In the liver—the most active site of lipid (cholesterol, fatty acids, and other lipid compounds) synthesis, the lipids made in the liver and those collected from chylomicron remnants are packaged with proteins as a VLDL (very-low-density lipoprotein) and shipped to other parts of the body. As the VLDL travel through the body, cells remove triglycerides. As they lose triglycerides, the VLDL shrink and the proportion of lipids shifts. Cholesterol becomes the predominant lipid, and the lipoprotein becomes smaller and more dense. As this occurs, the VLDL becomes an LDL (low-density lipoprotein), loaded with cholesterol, but containing relatively few triglycerides. HDL (high-density lipoprotein) remove cholesterol from the cells and carry it back to the liver for recycling or disposal. By efficiently clearing cholesterol, HDL lowers the risk of heart disease. In addition, HDL have anti-inflammatory properties that seem to keep artery-clogging plaque from breaking apart and causing heart attack. FATTY ACIDS BOUND TO HUMAN SERUM ALBUMIN § ~7 binding sites for fatty acids § Up to 2 fatty acids bound under normal physiological conditions § About 6 fatty acids bound during fasting… Simard, J. R. et al. (2005) PNAS 102, 17958-17963 STORED TRIACYLGLYCEROLS ARE MOBILIZED FROM LIPID DROPLETS BY HORMONES § A signal triggered by low blood glucose stimulates conversion of TGs to fatty acids by hormone-sensitive lipase, HSL (NOTE - example of regulation of enzyme activity by phosphorylation) § Fatty acids leave the adipocyte and are carried in the bloodstream by binding to serum albumin § They can be released from albumin to enter cells where they are oxidized to provide energy Mobilization of triacylglycerols stored in adipose tissue. When low levels of glucose in the blood trigger the release of glucagon, 1 the hormone binds its receptor in the adipocyte membrane and thus 2 stimulates adenylyl cyclase, via a G protein, to produce cAMP. This activates PKA, which phosphorylates 3 the hormone-sensitive lipase (HSL) and 4 perilipin molecules on the surface of the lipid droplet. Phosphorylation of perilipin causes 5 dissociation of the protein CGI from perilipin. CGI then associates with the enzyme adipose triacylglycerol lipase (ATGL), activating it. Active ATGL 6 converts triacylglycerols to diacylglycerols. The phosphorylated perilipin associates with phosphorylated HSL, allowing it access to the surface of the lipid droplet, where 7 it converts diacylglycerols to monoacylglycerols. A third lipase, monoacylglycerol lipase (MGL) 8, hydrolyzes monoacylglycerols. 9 Fatty acids leave the adipocyte, bind serum albumin in the blood, and are carried in the blood; they are released from the albumin and 10 enter a myocyte via a specific fatty acid transporter. 11 In the myocyte, fatty acids are oxidized to CO2, and the energy of oxidation is conserved in ATP, which fuels muscle contraction and other energy-requiring metabolism in the myocyte. HYDROLYSIS OF TRIACYLGLYCEROLS YIELDS FATTY ACIDS AND GLYCEROL Fats (TAGs) are degraded into fatty acids and glycerol in the cytoplasm of adipocytes Hydrolysis of triacylglycerols is catalyzed by lipases Free Fatty Acids; (FFA): contains ~95% of the energy of TAGs Glycerol contains ~5% of the energy of TAGs Some lipases are regulated by hormones glucagon and epinephrine (see HSL) Epinephrine means: “We need energy now” Glucagon means: “We are out of glucose” GLYCEROL FROM TAG ENTERS GLYCOLYSIS Glycerol kinase activates glycerol at the expense of ATP, predominantly in liver, to give glycerol-P Subsequent reactions (glycolysis then oxphos) recover more than enough ATP to cover this cost Note: Glycerol Kinase is absent in adipocytes, so that glycerol released from TAG degradation cannot be used in this tissue during starvation. Rather it is exported (through the blood) to liver where it can be converted to TAGs, or to DHAP (glycerol 3-P dehydrogenase) which enters gluconeogenesis FREE FATTY ACID ARE TRANSPORTED TO MITOCHONDRIA Fatty acids are transported to other tissues for fuel b-oxidation of fatty acids in these tissues occurs in mitochondria Small (less than12 carbons) fatty acids diffuse freely across mitochondrial membranes Larger fatty acids (most free fatty acids) are transported via acyl-carnitine/carnitine transporter TRANSPORT OF FATTY ACIDS REQUIRES CONVERSION TO FATTY ACYL-COA FA + CoA + ATP ßà FA-CoA + AMP + PPi Fatty acyl CoAs are high energy compounds Conversion of a fatty acid to a fatty acyl–CoA. The conversion is catalyzed by fatty acyl–CoA synthetase and inorganic pyrophosphatase. Fatty acid activation by formation of the fatty acyl–CoA derivative occurs in two steps. 2 ATP equivalents are used The overall reaction is highly exergonic (DG for hydrolysis ~ -34 kJ/mol). TRANSPORT OF FATTY ACYLS-CoA INTO MITOCHONDRIA BY THE CARNITINE SHUTTLE Fatty acyl-CoA esterified to -OH of carnitine CAT II CAT I Carnitine/acylcarnitine Fatty acid entry into mitochondria via the acyl-carnitine/carnitine transporter. After fatty acyl– carnitine is formed at the outer membrane or in the intermembrane space, it moves into the matrix by facilitated diffusion through the transporter in the inner membrane. In the matrix, the acyl group is transferred to mitochondrial coenzyme A, freeing carnitine to return to the intermembrane space through the same transporter. Acyltransferase I is inhibited by malonyl-CoA, the first intermediate in fatty acid synthesis. This inhibition prevents the simultaneous synthesis and degradation of fatty acids. ACYL-CARNITINE/CARNITINE TRANSPORT Transport of fatty acids into the mitochondrial matrix is rate-limiting for fatty acid oxidation and an important point of regulation Once fatty acids enter the mitochondria, they are committed to oxidation Carnitine deficiencies (dietary, genetic) leads to reduced ability to use long chain fatty acids as metabolic fuel FA less than12 carbons enter mitochondria as free acids and are converted to acyl CoA derivatives in mitochondrial matrix THE b-OXIDATION PATHWAY OF SATURATED FATTY ACIDS HAS FOUR BASIC STEPS The β-oxidation pathway. Each pass removes one acetyl moiety in the form of acetyl-CoA. 4 steps per cycle in which an acyl-CoA ester undergoes: § dehydrogenation § hydration § dehydrogenation § thiolytic cleavage § Each cycle produces an acetylCoA and shortens the chain by two carbons, giving 8 acetyl-CoA from one palmitoyl-CoA (a) In each pass through this fourstep sequence, one acetyl residue (shaded in pink) is removed in the form of acetyl-CoA from the carboxyl end of the fatty acyl chain—in this example palmitate (C16), which enters as palmitoyl-CoA. (b) Six more passes through the pathway yield seven more molecules of acetyl-CoA, the seventh arising from the last two carbon atoms of the 16-carbon chain. Eight molecules of acetyl-CoA are formed in all. u Steps 2, 3 and 4 for fatty acids longer than C12 are carried out by a multienzyme complex (trifunctional protein) in the mitochondrial inner membrane; (see later) u Shorter fatty acids are processed by soluble matrix enzymes STEP 1: DEHYDROGENATION OF ALKANE TO ALKENE Catalyzed by 3 isoforms of Acyl-CoA Dehydrogenase (AD) on the innermitochondrial membrane – Very-long-chain AD (12–18 carbons) – Medium-chain AD (6–14 carbons)* – Short-chain AD (4–8 carbons) Results in trans double bond, different from naturally occurring unsaturated fatty acids Analogous to succinate dehydrogenase reaction in the citric acid cycle – Electrons from bound FAD transferred directly to the electron- transport chain via Electron-Transferring Flavoprotein (ETF) Go to ETF Reaction: Deprotonation of Ca; then hydride transfer from Cb to FAD * MCAD deficiency (common:1 in 10,000): genetic defect in FA catabolism due to recessive mutation in MCAD leading to hypoketotic (defect in ketone bodies formation) hypoglycemia (cell rely on glucose for energy) and liver dysfunction. A REMINDER: PATH OF ELECTRONS FROM FATTY ACYL–COA TO UBIQUINONE. Acyl-CoA dehydrogenase (the first enzyme of β oxidation) transfers electrons to electron-transferring flavoprotein (ETF), from which they pass to Q via ETF: ubiquinone oxidoreductase. STEP 2: HYDRATION OF ALKENE Catalyzed by two isoforms of enoyl-CoA hydratase: – Soluble short-chain hydratase (crotonase family) – Membrane-bound long-chain hydratase, part of trifunctional complex (step 2, 3 and 4) – Acts on trans double bonds Water adds across the double bond yielding alcohol Analogous to fumarase reaction in the CAC – Same stereospecificity Acts on trans double bonds Reaction: water is added to the C2(Ca)-C3(Cb) double bond STEP 3: A SECOND DEHYDROGENATION STEP PRODUCES b-KETOACYL-CoA & NADH Catalyzed by two isoforms of bhydroxyacyl-CoA dehydrogenase – Soluble short-chain DHase (crotonase family) – Membrane-bound long-chain DHase, part of trifunctional complex (step 2, 3 and 4) The enzyme uses NAD cofactor as the hydride acceptor Only L-isomers of hydroxyacyl CoA act as substrates Analogous to malate dehydrogenase reaction in the citric acid cycle Go to complex 1 Reaction: dehydrogenation of alcohol STEP 4: THIOLASE SPLITS OFF ACETYL-CoA Catalyzed by acyl-CoA acetyltransferase (thiolase) via covalent mechanism (also two isoforms, one for short chain and one for long chain, part of Trifunctional protein) – The carbonyl carbon in bketoacyl-CoA is electrophilic – Active site thiolate acts as nucleophile and releases acetyl-CoA – Terminal sulfur in CoA-SH acts as nucleophile and picks up the fatty acid chain from the enzyme The net reaction is thiolysis of carbon-carbon bond THE 4-STEP CYCLE REPEATS: 8 ACETYL-CoAS FROM ONE PALMITOYL-CoA Each round produces an acetyl-CoA and shortens the chain by two carbons FATTY ACID CATABOLISM FOR ENERGY For palmitic acid (C16) – Repeating the above four-step process six more times (7 total) results in eight molecules of acetyl-CoA FADH2 is formed in each cycle (7 total) NADH is formed in each cycle (7 total) Acetyl-CoA enters citric acid cycle and further oxidizes into CO2 – This makes more GTP, NADH, and FADH2 Electrons from all FADH2 enter electrontransferring flavoprotein ETF and those from NADH enter Complex I TOTAL ATP YIELD OF OXYDATION OF ONE FATTY ACID OF 16 CARBONS - b-oxidation dehydrogenases: 7FADH2+7NADH (=28 ATP) - 8 Acetyl CoA oxidation in the TCA cycle: 80 ATP à 108 ATPs produced by oxidizing C16:0-CoA to CO2 and H2O FAO CAC Note: From palmitate: 106 ATPs, since 2 ATP equivalents are consumed in the activation of palmitate (ATP to ADP + PPi and then PPi in 2 Pi) OXIDATION OF PROPIONYL-COA Odd numbered fatty acid Same reaction as pyruvate carboxylase or acetylCoA carboxylase Deoxyadenosyl Cobalamine * Oxidation of propionyl-CoA produced by β oxidation of oddnumber fatty acids. - The sequence involves the carboxylation of propionyl-CoA to Dmethylmalonyl-CoA and conversion of the latter to succinyl-CoA. - This conversion requires epimerization of D- to Lmethylmalonyl-CoA, followed by a remarkable reaction in which substituents on adjacent carbon atoms exchange positions - Isomerization in propionate oxidation requires coenzyme B12 Note: conversion of propionyl-CoA to succinylCoA is also involved in the catabolism of certain ‘glucogenic’ amino acids such as isoleucine and valine (use also deoxyadenosyl cobalamine) CAC *The other form is methylcobalamine, used by methionine synthase in the synthesis of Met COMPLEX COBALT-CONTAINING COMPOUND: COENZYME B12 Coenzyme B12 is the cofactor form of vitamin B12, which is unique among all vitamins: -Contains a complex organic molecule -Contains an essential trace element, Cobalt Vitamin B12 (cyanocobalamin) contains a cyano group attached to Cobalt In 5’ deoxyadenosylcobalamin (cofactor of methylmalonyl CoA mutase), the cyano group is replaced by 5’ deoxyadenosyl group (red) In methyl cobalamin (cofactor of methionine synthase), the cyano group is replaced by a methyl group group (not shown) Vitamin B12 deficiency results in pernicious anemia CONVERSION OF KETONE BODIES TO ACETYL-CoA IN EXTRA-HEPATIC TISSUES Conditions that promote gluconeogenesis (untreated diabetes, severely reduced food intake): - slow the citric acid cycle (by drawing off oxaloacetate), - enhance the conversion of acetyl-CoA to acetoacetate. The released CoA from ketone bodies formation allows continued β oxidation of fatty acids. Thiophorase b-ketoacyl-CoA transferase Almost all tissues / cell types with the exception of liver and red blood cells are able to use ketone bodies as fuel; liver cannot convert acetoacetate to acetoacetyl-CoA (lacks thiophorase) & RBCs have no mitochondria FORMATION OF KETONE BODIES Healthy, well-nourished individuals produce ketone bodies at a relatively low rate. However, in diabetes or during starvation, oxaloacetate of the CAC cycle is depleted, and the accumulated acetyl-CoA is converted into ketone bodies (this frees CoA for continued β-oxidation) The first step of ketone bodies formation is reverse of the last step in the b-oxidation: thiolase reaction joins two acetyl-CoA units to form acetoacetyl-CoA the parent compound of ketone bodies. ketone body formation occurs in the matrix of liver mitochondria. Acetone is exhaled while D-βHydroxybutyrate is used as a fuel Reverse of last step of b-oxidation THE KETONE BODY D-b-HYDROXYBUTYRATE CAN BE USED AS A FUEL D-β-Hydroxybutyrate, synthesized in the liver, passes into the blood and thus to other tissues (organs other than liver can use ketone bodies as fuels), where it is converted in three steps to acetyl-CoA. D-β-Hydroxybutyrate is first oxidized to acetoacetate, which is activated with coenzyme A donated from succinyl-CoA, then split by thiolase. The acetyl-CoA thus formed is used for energy production. High levels of acetoacetate and bhydroxybutyrate lower blood pH dangerously (acidosis) Thiophorase CATABOLISM AND ANABOLISM OF FATTY ACIDS PROCEED VIA DIFFERENT PATHWAYS Catabolism (oxidation) of fatty acids – produces acetyl-CoA – produces reducing power (NADH) – takes place in the mitochondria Anabolism (synthesis) of fatty acids – requires acetyl-CoA and malonyl-CoA – requires reducing power from NADPH – takes place in cytosol in animals, chloroplast in plants CATABOLISM AND ANABOLISM OF FATTY ACIDS PROCEED VIA DIFFERENT PATHWAYS Catabolism (oxidation) of fatty acids – produces acetyl-CoA – produces reducing power (NADH) – takes place in the mitochondria Anabolism (synthesis) of fatty acids – requires acetyl-CoA and malonyl-CoA – requires reducing power from NADPH – takes place in cytosol in animals, chloroplast in plants OVERVIEW OF FATTY ACID SYNTHESIS Fatty acids are built in several passes, processing one acetate unit at a time (by the fatty acid synthase complex in the cytoplasm where NADPH/NADP is high). The acetate is coming from activated malonate in the form of malonyl-CoA (a 3-Carbons intermediate) (formation of malonate is catalyzed by Acyl CoA Carboxylase (ACC) Each pass involves reduction of a carbonyl carbon to a methylene carbon. Synthesis may be viewed as b-oxidation in reverse, but – uses different enzymes and cofactors – Takes place in a different subcellular location The end-product is palmitoyl-CoA which inhibits Acyl CoA Carboxylase COMPARISON OF STEPS IN FATTY ACID SYNTHESIS & β-OXIDATION β-oxidation SPLIT synthesis CONDENSATION OXIDATION REDUCTION HYDRATION DEHYDRATION OXIDATION REDUCTION ACETYL-COA IS TRANSPORTED FROM MITOCHONDRIA INTO THE CYTOSOL AS CITRATE Acetyl-CoA is made in the mitochondria But lipid synthesis occurs in the cytosol, and there is no way for acetyl-CoA to cross mitochondrial inner membrane to the cytosol So, acetyl-CoA is converted to citrate. – Acetyl-CoA + oxaloacetate à citrate (in mitochondria) Same reaction as that of the CAC Catalyzed by citrate synthase Citrate is then shuttled to the cytosol: Citrate passes through the citrate transporter CITRATE IN THE CYTOSOL IS CLEAVED TO REGENERATE ACETYL-COA Citrate (now in cytosol) is cleaved by citrate lyase – Regenerates acetyl-CoA and oxaloacetate (in cytosol) – The reaction requires 1 ATP – Acetyl-CoA can now be used to make malonyl-CoA for lipid synthesis – What happens to the cytoplasmic oxaloacetate? (there is no oxaloacetate transporter in mitochondria) OXALOACETATECYT IS CONVERTED TO MALATE, WHICH HAS TWO FATES Malate dehydrogenase in cytosol reduces oxaloacetate to malate Two potential fates for malate: – Can be converted to pyruvatecyt and NADPHcyt via the malic enzyme (major route) NADPH used for lipid synthesis Pyruvatecyt sent back to mitochondria via pyruvate transporter Pyruvate is converted back in mitochondria to oxaloacetatemt by pyruvate carboxylase, requiring 1 ATP – Can be transported back to mitochondria via Malate-a-ketoglutarate transporter Malatemt is then reoxidized to oxaloacetatemt SHUTTLE FOR TRANSFER OF ACETYL GROUPS FROM MITOCHONDRIA TO THE CYTOSOL The mitochondrial outer membrane is freely permeable to all these compounds. Pyruvate derived from amino acid catabolism in the mitochondrial matrix, or from glucose by glycolysis in the cytosol, is converted to acetyl-CoA in the matrix. Acetyl groups pass out of the mitochondrion as citrate; in the cytosol they are delivered as acetylCoA for fatty acid synthesis. Oxaloacetate is reduced to malate, which can return to the mitochondrial matrix and is converted to oxaloacetate. The major fate for cytosolic malate is oxidation by malic enzyme to generate cytosolic NADPH; the pyruvate produced returns to the mitochondrial matrix. ACETYL-CoACYT AND BICARBONATE FORM MALONYL-CoA Reaction carboxylates acetyl CoA Catalyzed by Acetyl-CoA Carboxylase (ACC) (rate limiting step of fatty acid synthesis) – Enzyme has three subunits: One unit has Biotin to carry CO2 HCO3− (bicarbonate) is the source of CO2 Uses one ATP molecule In animals, all three subunits are on one polypeptide chain Therefore, the cost of malonyl CoA formation (precursor for fatty Acid synthesis) is 3 ATPs per 2-carbons unit (2 for shuttling acetyl-CoA to the cytosol and 1 for making malonyl-CoA) A REMINDER: BIOTIN CARRIES CO2 Two-step reaction similar to carboxylations catalyzed by pyruvate carboxylase and propionyl-CoA carboxylase CO2 binds to biotin - Bicarbonate react with ATP to produce carboxy phosphate as an intermediate - Carboxyphosphate breaks down to CO2 - CO2 is attached to N in ring of biotin - Biotin transports CO2 to acetyl CoA on another active site THE ACETYL-COA CARBOXYLASE (ACC) REACTION Malonyl-CoA is formed from Acetyl-CoA and Bicarbonate ACA has 3 functional regions: § Biotin carrier protein (gray) § Biotin carboxylase, which activates CO2 by attaching it to a nitrogen in the biotin ring in an ATP-dependent reaction § Transcarboxylase, which transfers activated CO2 (shaded green) from biotin to acetyl-CoA, producing malonyl-CoA The long, flexible biotin arm carries the activated CO2 from the biotin carboxylase region to the transcarboxylase active site. The active enzyme in each step is shaded blue SYNTHESIS OF FATTY ACIDS IS CATALYZED BY FATTY ACID SYNTHASE (FAS) Catalyzes a repeating four-step sequence that elongates the fatty acyl chain by two carbons at each step – Uses NADPH as as the electron donor – Uses two enzyme-bound -SH groups as activating groups FAS I in vertebrates and fungi FAS II in plants and bacteria FATTY ACID SYNTHESIS OCCURS IN CELL COMPARTMENTS WHERE NADPH LEVELS ARE HIGH Cytosol for animals, yeast Chloroplast for plants Major sources of NADPH*: – In adipocytes: pentose phosphate pathway and malic enzyme NADPH made as malate converts to pyruvate + CO2 – In hepatocytes and mammary gland: pentose phosphate pathway NADPH made as glucose-6phosphate converts to ribulose 6phosphate – In plants: photosynthesis Two routes to NADPH, catalyzed by (a) malic enzyme (b) the pentose phosphate pathway * NADP dependent Isocitrate dehydrogenase to a lesser extent THE OVERALL PROCESS OF PALMITATE SYNTHESIS: SEVEN CYCLES OF CONDENSATION AND REDUCTION The fatty acyl chain grows by two-carbon units donated by activated malonate, with loss of CO2 at each step. The initial acetyl group is shaded yellow, C-1 and C-2 of malonate are shaded pink, and the carbon released as CO2 is shaded green. After each two-carbon addition, reductions convert the growing chain to a saturated fatty acid of four, then six, then eight carbons, and so on. The final product is palmitate (16:0). STOICHIOMETRY OF SYNTHESIS OF PALMITATE (16:0) 1. Formation of 7 malonyl CoA : 7 acetyl-CoAs are carboxylated to make 7 malonyl-CoAs… using ATP: 7 Acetyl-CoA + 7 CO2 + 7 ATP -> 7 Malonyl-CoA + 7 ADP + 7 Pi 2. 7 Cycles of condensation, reduction dehydration and reduction… using NADPH to reduce the b-keto group and trans-double bond: Acetyl-CoA + 7 Malonyl-CoA + 14 NADPH + 14 H+ -> Palmitate + 7 CO2 + 8 CoA + 14 NADP+ + 6 H2O 3. Sum: 8 Acetyl-CoA + 7 ATP + 14 NADPH + 14 H+ -> Palmitate + 8 CoA + 6 H2O + 7 ADP + 7 Pi + 14 NADP+ Note: Eukaryotes have one additional energy cost (the two ATP needed to shuttle acetyl CoA to the cytosol). Therefore, the cost of Fatty Acid synthesis is 3 ATPs per 2-Carbons unit (2 for shuttling acetyl-CoA to the cytosol and 1 for making malonyl-CoA) minus 1 ATP for the first acetyl CoA (which is not made from malonyl CoA). FATTY ACID SYNTHESIS IS TIGHTLY CONTROLLED VIA ALLOSTERIC REGULATION OF ACC Acetyl CoA carboxylase (ACC) catalyzes the rate-limiting step – ACC is feedback-inhibited by palmitoyl-CoA – ACC is activated by citrate Remember citrate is made from acetyl-CoAmt When [acetyl-CoA]mt increases (with concomitant increase in ATP), signaling energy excess, acetylCoA is converted to citrate…citrate is exported to cytosol, and ACC is activated. Thus, citrate signals excess energy to be converted to fat. Note: Citrate inhibits also glycolysis by inhibiting PFK-1 ACC IS ALSO HORMONALLY REGULATED BY COVALENT MODIFICATION ACC is inhibited when energy is needed (low blood glucose) through: – Glucagon and epinephrine: reduce sensitivity of citrate activation lead to phosphorylation and inactivation of ACC (phosphorylated monomers of ACC are inactive) ACC is activated when energy is plenty (high blood glucose) through: – Insulin: leads to dephosphorylation of ACC monomers which polymerize into long active ACC filaments REGULATION OF FATTY ACID SYNTHESIS IN VERTEBRATES Both allosteric regulation and hormone-dependent covalent modification influence the flow of precursors into malonyl-CoA Regulation of fatty acid synthesis: Allosteric Hormonal Insulin triggers dephosphorylation and activation (ACC polymerization) (ACC depolymerization) Active form of ACC (a) In the cells of vertebrates, both allosteric regulation and hormone-dependent covalent modification influence the flow of precursors into malonyl-CoA. (b) Filaments of acetyl-CoA carboxylase (the active, dephosphorylated form) as seen with the electron microscope. COORDINATED REGULATION OF FATTY ACIDS SYNTHESIS AND BREAKDOWN * *Glucagon also triggers mobilization of fatty acids from lipid droplets in adipose tissue (through phosphorylation of HSL) REGULATION TARGETS Synthesis: Acetyl-CoA Carboxylase (ACC) Oxidation: Carnitine Acyl Transferase I (CAT I) When the diet provides a ready source of carbohydrate as fuel, β oxidation of fatty acids is unnecessary and is therefore downregulated. Two enzymes are key to the coordination of fatty acid metabolism: acetyl-CoA carboxylase (ACC), the first enzyme in the synthesis of fatty acids), and carnitine acyltransferase I, which limits the transport of fatty acids into the mitochondrial matrix for β oxidation. Ingestion of a high-carbohydrate meal raises the blood glucose level and thus 1 triggers the release of insulin. 2 Insulin-dependent protein phosphatase dephosphorylates ACC, activating it. 3 ACC catalyzes the formation of malonyl-CoA (the first intermediate of fatty acid synthesis), and 4 malonyl-CoA inhibits carnitine acyltransferase I, thereby preventing fatty acid entry into the mitochondrial matrix. When blood glucose levels drop between meals, 5 glucagon release activates cAMP-dependent protein kinase (PKA), which 6 phosphorylates and inactivates ACC. The concentration of malonyl-CoA falls, the inhibition of fatty acid entry into mitochondria is relieved, and 7 fatty acids enter the mitochondrial matrix and 8 become the major fuel. Because glucagon also triggers the mobilization of fatty acids in adipose tissue, a supply of fatty acids begins arriving in the blood. Also, transcriptional regulation of CAT, acyl CoA dehydrogenases... PLANTS, BUT NOT MAMMALS, CAN DESATURATE POSITIONS BEYOND C-9 Humans have D4, D5, D6, and D9 desaturases but cannot desaturate beyond D9 Plants can produce: – linoleate 18:2(D9,12) – a-linolenate 18:3 (D9,12,15) These fatty acids are “essential” to humans – Polyunsaturated fatty acids (PUFAs) help control membrane fluidity – PUFAs are precursors to eicosanoids LONGER-CHAIN AND UNSATURATED FATTY ACIDS ARE SYNTHESIZED FROM PALMITATE Routes of synthesis of other fatty acids: Elongation of Palmitate (16:0) à stearate(18:0) – Catalyzed by elongase (works by adding two carbons at the COOH end, after activation, and using malonylCoA, just like FAS. (Elongases) (Desaturases) Desaturation of Palmitate (16:0) à palmitoleate (16:1; D9), Stearate (18:0) à oleate (18;1; D9): – Catalyzed by fatty acyl-CoA desaturase in animals – Requires NADPH; enzyme uses cytochrome b5 and cytochrome b5 reductase, see next slide. (Note that this is a D9-desaturase! It reduces the bond between C-9 and C-10). – Mammals cannot desaturate beyond, thus cannot convert oleate to linoleate or α-linolenate (shaded w3 precursor pink), which are therefore required in the diet as essential fatty acids. ALA – Conversion of a-linolenate (a-ALA, essential fatty acid) to other polyunsaturated fatty acids (EPA, 20:5(D5,8,11,14,17), DHA 22:6(D4,7,10,13,16,19) and eicosanoids by elongation/desaturation EPA, DHA is outlined. w6 precursor BIOSYNTHESIS OF TRIACYLGLYCEROLS 1/ Synthesis of Phosphatidic Acid 2/ Conversion of Phosphatidic from glycerol-P and acyl-CoAs Acid into Triacylglycerol Phosphatidic acid is the precursor of both TAGs and Glycerophospholipids Phosphatidic acid phosphatase (lipin) removes the 3phosphate from the phosphatidic acid – Yields 1,2diacylglycerol Third carbon is then acylated with a third fatty acid – Yields triacylglycerol BIOSYNTHESIS OF MEMBRANE LIPIDS PHOSPHODIESTER BOND OF PHOSPHOLIPIDS FROM CDP (See also next slide) 1/ DAG is activated with CDP 2/ the head group is transferred (Example: Inositol àphosphatidylinositol) 1/ Headgroup is activated with CDP 2/ DAG is transferred. (Example: CDPcholine à phosphatidylcholine) Two general strategies for forming the phosphodiester bond of phospholipids. In both cases, CDP supplies the phosphate group of the phosphodiester bond. SPHINGOLIPIDS ARE MADE IN FOUR STEPS 1) Synthesis of sphinganine from palmitoylCoA and serine 2) Attachment of fatty acid via amide linkage 3) Desaturation of sphinganine Yields N-acylsphingosine (ceramide) 4) Attachment of head group Can yield a cerebroside or ganglioside OVERVIEW OF AMINO ACID CATABOLISM used for § The amino groups and the carbon skeleton take separate but interconnected pathways: § The amino group enters the urea cycle and nitrogen is excreted in the form of urea excreted § The carbon skeleton enters the citric acid cycle to be oxidized AMINO GROUP CATABOLISM Ammonia is toxic Urea is far less toxic than ammonia and has very high solubility Uric acid is rather insoluble Excretion as paste allows the animals to conserve water unless reused, amino groups are channeled into a single excretory end product: Humans and great apes excrete both urea (from amino acids) and uric acid (from purines) THE AMINO GROUP IS TRANSFERRED TO a-KETOGLUTARATE FORMING L-GLUTAMATE Urea or synthesis Catalyzed by aminotransferases or transaminases: Uses the pyridoxal phosphate (PLP) cofactor Typically, a-ketoglutarate accepts amino groups and is converted into glutamate Glutamate acts therefore as a temporary storage of nitrogen (in its amino group) Glutamate can then donate the amino group to form urea for amino acid biosynthesis when needed The amino acid is converted to an a-ketoacid TRANSAMINASES Cells contain many different Transaminases (aminotransferases). They are specific for α-ketoglutarate as the amino group acceptor, but differ in their specificity for the amino group donor. Named for the amino group donor: For instance, alanine aminotransferase will transfer an amino group from alanine to α-ketoglutarate to form pyruvate and glutamate. Aspartate aminotransferase will transfer an amino group from aspartic acid to α-ketoglutarate to form glutamate and oxaloacetate. The reactions catalyzed by transaminases are reversible. All amino transferases use pyridoxal phosphate (PLP) as a prosthetic group. This is the coenzyme form of pyridoxine, or vitamin B6. Pyridoxal phosphate is tightly bound to the transaminases active site through a Schiff-base (covalent) and other non-covalent interactions. IN THE LIVER, AMMONIA COLLECTED IN GLUTAMATE IS REMOVED BY GLUTAMATE DEHYDROGENASE (GDH) TO BE EXCRETED AS UREA In hepatocytes (Liver), Glutamate is transported from the cytosol to mitochondria and is deaminated by Transamination oxidative deamination within mitochondrial matrix, forming aketoglutarate GDH can use either NAD+ or NADP+ as electron acceptor Ammonia is processed into urea for excretion α-ketoglutarate can enter the citric acid cycle or be used for glucose synthesis Thus, the pathway for ammonia excretion is a transdeamination = transamination (transaminases) + oxidative deamination (GDH) Urea à excretion GLUTAMATE DEHYDROGENASE OPERATES AT AN IMPORTANT INTERSECTION IN CARBON AND NITROGEN METABOLISM α-ketoglutarate product can be oxidized as fuel (in CAC) or serve as a glucose precursor in gluconeogenesis (through oxaloacetate) glutamate dehydrogenase is: – positively modulated by ADP (signals low glucose levels) – negatively modulated by GTP (signals high levels of α-ketoglutarate) IN THE MUSCLE, AMMONIA COLLECTED IN GLUTAMATE IS DONATED TO PYRUVATE TO MAKE ALANINE Vigorously working muscles rely on glycolysis for energy: à yields pyruvate and operate nearly anaerobically if pyruvate is not eliminated lactic acid will build up To avoid lactic acid build-up, this pyruvate can be converted to alanine (by taking up the amino group from glutamate) for transport into liver: Once in the liver, alanine releases its amino group and becomes pyruvate, which is used to produce glucose. Glucose returns to the muscle and the ammonia is excreted (see the glucose-alanine cycle). ALANINE TRANSPORTS AMMONIA FROM SKELETAL MUSCLE TO THE LIVER: THE GLUCOSE-ALANINE CYCLE GDH § Alanine plays a special role in transporting amino groups to the liver via the glucose-alanine cycle. § In this cycle, the amino group of aminoacids collected in glutamate is transferred to pyruvate, using alanine aminotransferase (an alternative to the formation of glutamine via the glutamine synthetase reaction). § The alanine formed in this reaction can then be transported to the liver, where transamination once again produces pyruvate and glutamate § The pyruvate can then be used to synthesize glucose, via gluconeogenesis, which is exported back to the muscles. (Recall, lactic acid formed during vigorous activity is also exported to the liver for gluconeogenesis by the Cori Cycle). § The ammonia formed by deamination of glutamate (GDH reaction) is excreted. AMMONIA FROM OTHER TISSUES IS SAFELY TRANSPORTED TO THE LIVER IN THE BLOODSTREAM AS GLUTAMINE § Many tissues, including brain, generate free ammonia and in order to prevent the accumulation of high levels of this toxic substance, it must be converted into a non-toxic form for transport. § Excess ammonia in extrahepatic tissues is added to glutamate to form glutamine (non-toxic), a process catalyzed by glutamine synthetase (requires ATP). § After transport in the bloodstream, the glutamine enters the liver mitochondria where glutaminase, hydrolyzes the ammonia from the side chain of glutamine, converting back glutamine to glutamate. § NH4+ thus liberated is incorporated in urea then excreted (excess glutamine is processed in intestines, kidneys, and liver). excretion AMMONIA IS TOXIC TO ANIMALS Free ammonia is toxic, especially to the brain NH4+ competes with K+ for transport into astrocyte cells through Na+K+ ATPase – Leads to elevated extracellular [K+] Na+-K+-2Cl- cotransporter 1 (NKCC1) = symporter that transports Na+, K+, and Cl– excess Cl- from the excess K+ alters neuronal response to the neurotransmitter GABA HIGHLY TOXIC AMMONIA MUST BE UTILIZED OR EXCRETED IN UREA Urea contains two amino groups The first amino group of urea comes from free ammonia (released from glutamate or glutamine). This is done through Carbamoyl phosphate synthase I which captures the free ammonia in the mitochondrial matrix First step of the urea cycle This step is regulated The second amino group of urea is acquired from aspartate THE FIRST NITROGEN-ACQUIRING REACTION OF THE UREA CYCLE (Synthesis of carbamoyl phosphate from free ammonia) 1 1 This reaction uses 2 ATPs Nitrogen of carbamylphosphate enters the urea cycle In this first nitrogen-acquiring reaction, catalyzed by carbamoyl phosphate synthetase I, the first nitrogen enters from ammonia. The terminal phosphate groups of two molecules of ATP are used to form one molecule of carbamoyl phosphate. This reaction has 2 activation steps (1 and 3). THE SECOND NITROGEN-ACQUIRING REACTION OF THE UREA CYCLE (Entry of aspartate into the urea cycle) This reaction uses 2 ATPs Nitrogen of arginino-succinate enters in urea composition In this second nitrogen-acquiring reaction, catalyzed by argininosuccinate synthetase, the second nitrogen enters from aspartate. Activation of the ureido oxygen of citrulline in step 1, using 1 ATP and releasing PPi, sets up the addition of aspartate in step 2. THE AMINO GROUP OF GLUTAMATE OR GLUTAMINE IS METABOLIZED IN THE MITOCHONDRIA OF HEPATOCYTES Free ammonia (NH4+) released from glutamate (or glutamine) is converted to urea for excretion. Carbamoyl phosphate synthase I captures free ammonia in the mitochondrial matrix First step of the urea cycle Regulated One amino group of urea (from glutamate or glutamine) enters the urea cycle as carbamoyl phosphate, formed in the matrix; the other enters as aspartate, formed in the matrix by transamination of oxaloacetate and glutamate, catalyzed by aspartate aminotransferase. Urea cycle reactions Urea THE REACTIONS IN THE UREA CYCLE Urea cycle and reactions that feed amino groups into the cycle. The enzymes catalyzing these reactions are distributed between the mitochondrial matrix and the cytosol. One amino group enters the urea cycle as carbamoyl phosphate, formed in the matrix; the other enters as aspartate, formed in the matrix by transamination of oxaloacetate and glutamate, catalyzed by aspartate aminotransferase. The urea cycle consists of four steps (3 are irreversible): 1 Formation of citrulline from ornithine and carbamoyl phosphate (entry of the first amino group); the citrulline passes into the cytosol (Ornitine Transcarbamylase) 2 Formation of argininosuccinate through a citrullyl-AMP intermediate (entry of the second amino group) (Arginosuccinate synthase). 3 Formation of arginine from argininosuccinate; this reaction releases fumarate, which enters the citric acid cycle (Arginosuccinase). Only reversible step. 4 Formation of urea; this reaction also regenerates ornithine (Arginase). ATP COST OF THE UREA CYCLE § Urea is produced from ammonia in five enzymatic steps and requires 4 molecules of ATP. § Two molecules are required to form carbamoyl phosphate and the equivalent of two further ones are required in the formation of argininosuccinate. (ATP to AMP + PPi then PPi to 2 Pi has the same energy cost as the hydrolysis of 2 molecules of ATP to ADP + Pi). to malate and CAC § Some of the energy cost is made up by converting fumarate to malate (see next slide). The rest comes from GDH reaction (see next slide) THE UREA CYCLE AND CITRIC ACID CYCLE ARE LINKED THROUGH THE ASPARTATE-ARGINOSUCCINATE SHUNT The interconnected, urea and citric acid, cycles have been called the "Krebs bicycle." NH4+ Glutamate Makes 2.5 ATP Glutamate DH reaction makes 2.5 ATP The aspartate-argininosuccinate shunt (linking the citric acid and urea cycles), effectively link the fates of the amino groups and the carbon skeletons of amino acids. § In converting fumarate to malate, then malate to oxaloacetate, one molecule of NADH is generated, whose re-oxidation through the oxidative phosphorylation process generates 2.5 ATP. Thus the energy cost of the urea cycle is 1.5 ATP. § However, the glutamate dehydrogenase reaction that gives the ammonia to the urea cycle (see above) leads to the formation of one NADH and thus to 2.5 ATP molecules. § Thus, the urea cycle can be considered as The interconnections are even more elaborate than the arrows suggest. For example, some citric acid self-sustaining cycle enzymes, such as fumarase and malate dehydrogenase, have both cytosolic and mitochondrial isozymes. Fumarate produced in the cytosol—whether by the urea cycle, purine biosynthesis, or other processes—can be converted to cytosolic malate, which is used in the cytosol or transported into mitochondria (via the malate-aspartate shuttle) to enter the citric acid cycle. REVIEW: FLOW OF NITROGEN FROM AMINO ACIDS TO UREA Stoichiometry of the urea cycle Aspartate + NH3 + CO2 + 3 ATP --> Urea + fumarate + 2 ADP + 2 Pi + AMP + PPi + 3H20 PPi -> 2Pi (1 ATP equivalent) Urea is mainly excreted in the urine; some enters the intestine where bacterial urease cleaves it to CO2 and NH3 (which is lost in the feces and partly reabsorbed into the blood) When liver function is compromised or due to genetic defects in the urea cycle à Hyperammonemia (>>10 μM), NH3 is neurotoxic for reasons that are not clear but could include: - consumption of glutamate and ATP to make glutamine; - depletion of TCA cycle intermediate α-ketoglutarate to make glutamate; - neurotransmitter function of glutamate à GABA REGULATION OF THE UREA CYCLE BY N-ACETYL-GLUTAMATE N-acetylglutamate is formed by Nacetylglutamate synthase – When glutamate and acetyl-CoA concentrations are high (indicating the presence of high levels of aminoacids) N-acetylglutamate activates carbamylphosphate synthetase I, thus activating the urea cycle Also, high levels of arginine activate Nacetylglutamate synthase, making more N-acetylglutamate, which activates the cycle Gene expression of urea cycle enzymes increases when needed: – High protein diet – Starvation, when protein is being broken down for energy GENETIC DEFECTS IN THE UREA CYCLE CAN BE LIFE-THREATENING (JC11) § Individuals with genetic defects in any enzyme involved in urea formation cannot tolerate protein-rich diets. § Because amino acids ingested in excess of the daily requirements for protein synthesis are deaminated in liver, producing ammonia that cannot be converted to urea and transported into the bloodstream for excretion (ammonia is highly toxic). § Deficiency of a urea cycle enzyme can produce hyperammonemia and the build-up of one or more urea cycle intermediates. Most of the the urea cycle steps are irreversible, which means that the defect can often be diagnosed by determining which intermediate is present in particularly high concentrations in blood and/or urine. § Although the breakdown of amino acids can have serious health effects in individuals with urea cycle enzyme deficiencies, a protein-free diet is not an option. Humans are incapable of synthesizing nearly half of the amino acids and these must be provided in the diet. Such amino acids are called essential, while other are conditionally essential. JC11: GENETIC DEFECTS IN THE UREA CYCLE POSSIBLE TREATMENTS OF DEFECTS IN THE UREA CYCLE ENZYMES § The administration in the diet of phenylbutyrate can lower the level of ammonia in the blood by promoting urinary excretion of glutamine as phenylacetylglutamine. § The administration in the diet of benzoate (not shown) can lower the level of ammonia in the blood by promoting urinary excretion of glycine as benzoylglycine (hippurate). MECHANISM OF ACTION OF PHENYLBUTYRATE AND BENZOATE The administration in the diet of benzoate or phenylbutyrate can lower the level of ammonia in the blood by promoting urinary excretion of glycine as benzoylglycine (hippurate) and glutamine as phenylacetylglutamine. Benzoate is initially converted to benzoyl-CoA at the expenditure of ATP. In a second step, benzoyl-CoA reacts with glycine to form benzoylglycine (hippurate) that is then excreted in the urine. The glycine used in this process must be regenerated and ammonia is thus removed. Phenylbutyrate, administered in the diet, undergoes a round of β-oxidation, and leads to phenylacetate which is converted to phenylacetyl-CoA at the expense of ATP. Phenylacetyl-CoA reacts with glutamine to form phenylacetylglutamine which can be excreted. Subsequent synthesis of glycine and glutamine to replenish the pool of these intermediates removes ammonia from the bloodstream. Severe hyperammonemia is treated by hemodialysis Other therapies are more specific to a particular enzyme deficiency: - A deficiency in N-acetylglutamate synthetase results in the absence of the normal activator, N-acetylglutamate. This condition can be treated by administering an analogue of N-acetylglutamate, carbamoyl-glutamate (activates carbamoyl phosphate synthetase I). - Arginine is used for example to treat deficiencies of enzymes 1, 2 and 3 of the cycle (can pick up ammonia from CP or Aspartate). END PRODUCTS OF AMINO ACID DEGRADATION Intermediates of the Central Metabolic Pathway Some amino acids result in more than one intermediate Ketogenic amino acids can be converted to ketone bodies Seven to Acetyl-CoA Leu, Ile, Thr, Lys, Phe, Tyr, Trp Glucogenic amino acids can be converted to glucose Six to pyruvate Ala, Cys, Gly, Ser, Thr, Trp Five to a-ketoglutarate Arg, Glu, Gln, His, Pro Four to succinyl-CoA Ile, Met, Thr, Val Two to fumarate Phe, Tyr Two to oxaloacetate Asp, Asn SOME AMINO ACID ARE CONVERTED TO GLUCOSE, OTHERS IN KETONE BODIES Ketogenic amino acids: Seven amino acids are degraded, at least in part, to acetoacetyl-CoA or acetyl-CoA and therefore a source of potential ketone bodies. Their ability to form ketone bodies is particularly important in untreated diabetes in which the liver forms large amounts of ketone bodies. These seven amino acids are: phenylalanine, tyrosine, tryptophan, isoleucine,leucine, lysine and threonine. Glucogenic amino acids: All amino acids that can be degraded, at least in part, to either pyruvate, αketoglutarate, succinyl-CoA, fumarate and/or oxaloacetate can be converted to glucose via the gluconeogenesis pathway. The division between ketogenic and glucogenic is not absolute. Five amino acids are both ketogenic and glucogenic. These are tryptophan, phenylalanine, tyrosine, threonine and isoleucine. Amino acids are grouped according to their major degradative end product. In fact, only two amino acids, leucine and lysine, are entirely ketogenic. Of these two, leucine is very common in proteins and makes a substantial contribution to ketosis under starvation conditions. SEVERAL COFACTORS ARE INVOLVED IN AMINO ACID CATABOLISM Important in one-carbon transfer reactions Tetrahydrafolate (THF): formed from folate, an essential vitamin by dihydrofolate reductase. THF has different forms and transfers 1-carbon in different oxidation states (in the form of CH3, CH2OH, and CHO). Carbon generally comes from serine. Forms interconverted on THF before use S-adenosylmethionine (adoMet): is the preferred cofactor for methyl transfer in biological reactions (methyl from adoMet is 1000 times more reactive than THF methyl group) Synthesized from ATP and methionine Regeneration of adoMet uses N5-methyl THF (the only known use in mammals) Biotin (transfers CO2) TETRAHYDROFOLATE CYCLE Folate trap: Methionine synthase reaction regenerates THF from N5MTHF. If deficient, folates are trapped in the N5-MTHF form (see folates trap, next slides). Methionine synthase (THF) Conversions of one-carbon units on tetrahydrofolate: There are two entry points for one-carbon units into the H4 folate one-carbon pool. - One involves the removal of a hydroxymethyl group from serine, forming glycine and N5,N10-methylene H4folate. - The other involves formic acid, that can be utilized, at the expense of ATP to form initially N10-formyl H4folate and subsequently, N5, N10-methenyl-H4folate. Regardless of the entry point into the one carbon pool, the one carbon units may be oxidized and reduced to three different levels of oxidation state: - In its most reduced form (N5-methyl H4folate), the cofactor carries a methyl group; - in its intermediate state (N5,N10-methylene H4folate) the cofactor carries a methylene group (transferred as –CH2OH); and - in its most oxidized form a methenyl group 5 10 (N ,N -methenyl H4folate) (transferred either as –CHO, or – CH=NH). The primary source of one-carbon units for H4folate is conversion of serine to glycine producing N5,N10-methylene H4folate. Although, H4folate can carry a methyl group, its potential for transferring this group in biosynthetic reactions is insufficient for most biological reactions. What is required is a more potent donor of methyl groups in most biosynthetic reactions and that is S-adenosylmethionine. ADOMET IS BETTER THAN THF AT TRANSFERRING CH3 S-adenosylmethionine (adoMet) is the prefered cofactor for methyl transfer in biological reactions – Methyl from adoMet is 1000 times more reactive than THF methyl group Synthesized from ATP and methionine Regeneration uses N5-methyl THF – The only known use in mammals REGENERATION OF ADOMET: THE ACTIVATED METHYL CYCLE Regeneration of different forms of THF Folate trap: if methylcobalamin methylcobalamin (CoEnz B12) is not available, or if methionine synthase is deficient, the folates become trapped as N5-methyl THF and cannot regenerate the other forms of THF: Methyl transfer Synthesis of methionine and S-adenosylmethionine in an activated-methyl cycle. In the methionine synthase reaction (step 4), the methyl group is transferred to cobalamin to form methylcobalamin, which in turn is the methyl donor in the formation of methionine. S-Adenosylmethionine, which has a positively charged sulfur (and is thus a sulfonium ion), is a powerful methylating agent in several biosynthetic reactions. The methyl group acceptor (step 2) is designated R. THE LINK BETWEEN TWO VITAMINS: VITAMIN B12 AND FOLATE Vitamin B12 and folate are closely linked in the pathway to regenerate methionine If vitamin B12 is not available à No methyl-cobalamin for the methionine synthase reaction), à à the folates become trapped as N5-methyl THF (folate trap) and cannot regenerate THF from N5methyl THF. If THF is not regenerated, N5,N10-methylene THF, which is used in DNA synthesis (through thymidylate synthase reaction that makes dTMP), is not formed (see folates cycle) à à Megaloblastic anemia (manifests as a decline in mature erythrocytes which are slowly replaced by smaller numbers of abnormally large erythrocytes called macrocytes, since cells cannot divide). This anemia can be partially treated with folates (THF), since Vitamin B12 deficiency alone will not cause the syndrome in the presence of sufficient folate (the mechanism is loss of B12 -dependent folate recycling needed for DNA synthesis). If vitamin B12 is not available à No methyl-cobalamin for the methionine synthase reaction à à No methionine formation à (no methyl transfer (since no adomet), no cysteine à oxidative stress etc.) Accumulation of homocysteine à homocysteinemia and homocysteinuria (rise in homocysteine in blood and urine) à (heart disease, hypertension and stroke). High levels of homocysteine may be responsible for 10% of all cases of heart disease. If vitamin B12 is not available à No deoxyadenosyl-cobalamin for the methyl malonyl CoA mutase reaction à à no degradation, accumulation of odd carbon numbered fatty acid à neurological disorders Rare defect in intestinal absorption of vitamin B12 (no Intrinsic Factor) or in vegetarians (B12 does not occur in plants) à Pernicious anemia and neurologic disorders: The anemia symptoms of pernicious anemia can be treated by administering either vitamin B12 or folate. However, the neurological symptoms of pernicious anemia cannot be treated by folate alone, because these symptoms result not from a defect in the methionine synthase reaction but from an accumulation of odd-carbon numbered fatty acids in neural membranes (role of deoxyadenosylcobalamin, in the conversion of methylmalonyl-CoA to succinyl-CoA ). BRANCHED CHAIN AMINO ACIDS METABOLISM DISEASE In a relatively rare genetic disease, the α-keto acid dehydrogenase complex is defective leading to an accumulation of the precursor amino acids and the three branched-chain α-keto acids in the blood and urine. This condition is called maple syrup urine disease, because of the characteristic odor of the urine because of the branched keto acids. Untreated, this disease results in abnormal development of the brain, mental retardation and death early in infancy. Treatment entails rigid control of diet, limiting the intake of valine, leucine and isoleucine to the minimum required to permit normal growth. BRANCHED CHAIN AMINO ACIDS ARE NOT DEGRADED IN THE LIVER Odd number Fatty acid degradation Propionyl CoA Coenzyme B12 (adenosyl form) Succinyl CoA Absent in the liver Methyl-crotonyl CoA carboxylase (Biotin) Acetyl CoA Most amino acids are catabolized in the liver, however, the three branched amino acids, valine, leucine and isoleucine are oxidized as fuels largely in muscle, adipose tissue, kidney and brain. These tissues contain a branched-chain aminotransferase that is absent in the liver. In a second reaction, a branched-chain α-keto acid dehydrogenase complex catalyzes oxidative decarboxylation of all three α-keto acids releasing the carboxyl group as CO2 and producing the acyl-CoA derivative. This reaction is analogous to the pyruvate dehydrogenase-catalyzed reaction that requires five cofactors (TPP, lipoate, CoASH, FAD, NAD). GENETIC DEFECTS IN MANY STEPS OF PHE DEGRADATION LEAD TO DISEASE In humans, these amino acids are normally converted to acetoacetyl-CoA and fumarate. Genetic defects in many of these enzymes cause inheritable human diseases (shaded yellow). ALTERNATIVE PATHWAYS FOR CATABOLISM OF PHENYLALANINE IN PHENYLKETONURIA (PKU) In individuals with a defect in phenylalanine hydroxylase, the level of phenylalanine rises, bringing into action a normally little used pathway. In this pathway, phenylalanine undergoes transamination to phenylpyruvate. Phenylalanine and phenylpyruvate accumulate in the blood and tissues and are excreted in the urine – hence the name phenylketonuria. Phenylpyruvate is decarboxylated to phenylacetate or reduced to phenyllactate (may also be found in the urine). The accumulation of phenylalanine or its metabolites early in life causes severe mental retardation. Mental retardation can be prevented by strict dietary control of phenylalanine intake. ANOTHER CAUSE OF PKU Role of tetrahydrobiopterin in the phenylalanine hydroxylase reaction. The H atom shaded pink is transferred directly from C-4 to C-3 in the reaction. Phenlyketonuria is also caused by a defect in the enzyme that regenerates the cofactor tetrahydrobiopterin, dihydrobiopterin reductase. In this case, however, the treatment is more complex than restricting the intake of Phe or Tyr, because tetrahydrobiopterin is required as a cofactor for other reactions that are precursors of the neurotransmitters norepinephrine and serotonin (so precursors such as L-dopa and 5-hydroxy-Trp must be supllied in the diet. Providing the cofactor itself is not effective because it cannot cross the blood-brain barrier. ALKAPTONURIA IS ANOTHER HERITABLE DISEASE OF PHENYLALANINE METABOLISM. In this case the defective enzyme is homogentisate dioxygenase. The effects of this defect are less serious, but individuals with this disease are likely to develop a form of arthritis. The excretion of large concentrations of homogentisate causes urine to turn black when the former is oxidized. JC11: SOME HUMAN GENETIC DISORDERS AFFECTING AMINO ACID CATABOLISM BIOSYNTHESIS OF NONESSENTIAL AMINO ACIDS IN HUMANS § Alanine (from pyruvate; transamination from glutamate) § Aspartate (from oxaloacetate; transamination from glutamate) § Asparagine (amidation of Asp; glutamine donates NH4+) § Glutamate (from α-ketoglutarate) § Serine (from 3-phosphoglycerate) DE NOVO BIOSYNTHESIS OF PURINES BEGINS WITH PRPP Adenine and guanine are synthesized as AMP and GMP from IMP Synthesis begins with the reaction of 5phosphoribosyl 1-pyrophosphate (PRPP) with Glutamine Purine ring builds up following addition of three carbons from glycine The first intermediate with full purine ring is inosinate (IMP) – Thus, PRPP + Gln à à à…IMP Ribose 5P ATP PRPP Synthase AMP SYNTHESIS OF IMP * *committed step A common pathway of 11 steps starting with phosphorybosylpyrophosphate (PRPP) and glutamine and ending to inosine monophosphate (IMP, also called or inosinate). The purine ring is synthesized while attached to ribose 5-P. Assembly of the ring continues one atom at a time until it reaches IMP. Formation of 5-phosphoribosylamine (step 1), catalyzed by glutamine-PRPP amidotransferase is the committed step in purine synthesis. (target of regulation). Step 6a occurs in higher eukaryotes FROM IMP, SYNTHESIS OF AMP AND GMP Feedback Inhibition Feedback Inhibition q The purine base in IMP is called hypoxanthine q Conversion of IMP to AMP requires the insertion of an amino group from aspartate. GTP is the source of energy q GMP is formed by the NAD+ oxidation of IMP, and ATP is used in the final step REGULATION OF PURINE BIOSYNTHESIS IS LARGELY FEEDBACK INHIBITION Four major mechanisms 1. Glutamine-PRPP amidotransferase (committed step of the pathway) is feedback inhibited by end-products IMP, AMP, and GMP 2. a/ Excess GMP feedback inhibits formation of xanthylate (XMP) from inosinate by IMP dehydrogenase, thus limiting its own formation, b/ Excess adenylate (AMP) feedback inhibits formation of adenylosuccinate by adenylosuccinate synthetase, thus limiting its own formation 3. GTP limits conversion of IMP to AMP, and ATP limits conversion of IMP to GMP 4. PRPP synthesis is inhibited by ADP and GDP DE NOVO PURINE NUCLEOTIDE SYNTHESIS IS REGULATED BY FEEDBACK INHIBITION *First step is not the committed step GDP 4 major feedback mechanisms cooperate: Pi *committed step PRPP 1/ Inhibition of the first reaction unique to purine synthesis, formation of 5-phosphoribosylamine (allosteric Glutamine-PRPP amidotransferase). 2/ At a later stage: Branch point. Inhibition of the formation of xanthylate (XMP) without affecting that of AMP and inhibition of formation of andenylosuccinate without affecting that of GMP. 3/ GTP required for synthesis of AMP while ATP is required for the synthesis of GMP = reciprocal control to balance the synthesis of the 2 nucleotides (see previous slides). 4/ Inhibition of PRPP synthesis by allosteric regulation of PRPP synthase by ADP and GDP Regulatory mechanisms in the biosynthesis of adenine and guanine nucleotides in mammals. See p292-295 Lippincott * PRPP can have other fates (salvage pathway, pyrimidines, NAD, NADP biosynthesis) à thus the step leading to its formation by PRPP synthetase is not the committed step. PYRIMIDINES ARE MADE FROM ASP, PRPP, AND CARBAMOYL PHOSPHATE Unlike purine synthesis, pyrimidine synthesis proceeds by first making the pyrimidine ring and then attaching it to ribose 5-phosphate First committed step is the reaction between Asp and carbamoylphosphate, catalyzed by aspartate transcarbamoylase (ATCase) DE NOVO SYNTHESIS OF PYRIMIDINE NUCLEOTIDES Glutamine + HCO3-+2ATP carbamoyl phosphate synthetase II Commited step CAD Biosynthesis of UTP and CTP via orotidylate. The pyrimidine is constructed from carbamoyl phosphate and aspartate (after carbamoyl phosphate, all the other atoms of the pyrimidine ring come from aspartate. The ribose 5-phosphate is then added to the completed pyrimidine ring by orotate phosphoribosyltransferase. UMP synthase OMP UMP Synthase In eukaryotes In prokaryotes The first step in this pathway (shown in red) is the synthesis of carbamoyl phosphate from CO2 and NH4+ from glutamine, catalyzed in eukaryotes by cytosolic carbamoyl phosphate synthetase II (part of CAD multifunctional complex*). This reaction resembles the first step of the urea cycle (catalyzed by carbamoyl phosphate synthetase I (in mitochondria), except that in this case, Glutamine is the nitrogen donor instead of NH4+ (in the case of the urea cycle). *In humans, the first 3 enzymes are a single multifunctional enzyme called CAD. OPRTase and ODCase activities are a single multifunctional enzyme called UMP synthase. UTP is the feedback inhibitor of CAD (while in prokaryotes CTP is the feedback inhibitor of ATCase) RIBONUCLEOTIDES DIPHOSPHATES ARE PRECURSORS OF DEOXYRIBONUCLEOTIDES DIPHOSPHATES 2’C-OH bond is directly reduced to 2’-H bond (ADPàdADP, GDPàdGDP, CDPàdCDP, UDPàdUDP)…without activating the carbon! (no analogous reactions are known) – Catalyzed by ribonucleotide reductase Mechanism: Two H atoms are donated by NADPH and carried by proteins thioredoxin or glutaredoxin dTMP IS MADE FROM dUTP (dUTPase) and dUMP (Thymidylate synthase) 1. dUTP is made (via deamination of dCTP or by phosphorylaton of dUDP) 2. dUTP à to dUMP by dUTPase 3. dUMP à dTMP by thymidylate synthase - adds a methyl group from tetrahydrofolate (Recall Folate trap and megaloblastic anemia) Thymidylate synthase is a target for some anticancer drugs. Biosynthesis of thymidylate (dTMP). DNA contains Thymine rather than Uracil and the de novo pathway to Thymine involves only deoxyribonucleotides. CONVERSION OF dUMP TO dTMP Thymidylate synthase: dUMP à dTMP (uses N5-N10 THF) Dihydrofolate reductase regenerates THF Serine hydroxymethyltransferase is required for regeneration of the N5,N10-methylene form of tetrahydrofolate (Recall also Folate trap à THF is not regenerated à megaloblastic anemia). In the synthesis of dTMP, all three hydrogens of the added methyl group are derived from N5,N10methyleneTHF. FOLIC ACID DEFICIENCY LEADS TO REDUCED THYMIDYLATE SYNTHESIS Folic acid* deficiency is widespread, especially in nutritionally poor populations (àheart disease, brain dysfunction and cancer) Reduced thymidylate synthesis leads to abnormal uracil incorporation into DNA DNA Repair mechanisms remove the uracil by creating strand breaks that affect the structure and function of DNA à cancer, heart disease, neurological impairment… Folates deficiency plays also a role in spinal bifida, a birth defect: incomplete formation of the spine and spinal cord during pregnancy MANY CHEMOTHERAPEUTIC AGENTS TARGET NUCLEOTIDE BIOSYNTHESIS Glutamine analogs: azaserine, acivicin – Inhibit glutamine amidotransferases (first enzyme of the de novo purine biosynthesis pathway) Fluorouracil – Converted by salvage pathway into FdUMP, which inhibits thymidylate synthase Methotrexate, trimethoprim and aminopterin – Inhibit dihydrofolate reductase (competitive inhibitors) CHEMOTHERAPY TARGETS: THYMIDYLATE SYNTHESIS AND FOLATE METABOLISM Fluorouracil and methotrexate are important chemotherapeutic agents. In cells, fluorouracil is converted to FdUMP, which inhibits thymidylate synthase. Methotrexate, a structural analog of tetrahydrofolate, inhibits dihydrofolate reductase; Trimethoprim, a tight-binding inhibitor of bacterial dihydrofolate reductase, was developed as an antibiotic. Fluorouracil AZASERINE AND ACIVICIN, INHIBITORS OF GLUTAMINE AMIDOTRANSFERASES These analogs of glutamine interfere in several amino acid and nucleotide biosynthetic pathways. CATABOLISM OF PURINES: FORMATION OF URIC ACID Purine ring is not cleaved and is excreted as poorly soluble uric acid Degradation of purines proceeds through dephosphorylation (via 5’-nucleotidase) – Adenosine is deaminated to inosine and then hydrolyzed to hypoxanthine and ribose – Guanosine yields xanthine via these hydrolysis and deamination reactions – Hypoxanthine and xanthine are then oxidized into uric acid by xanthine oxidase CATABOLISM OF PURINES: FORMATION OF URIC ACID excreted Adenosine deaminase Deficiency (ADA) leads to 100 fold increase of dATP, a strong inhibitor of ribonucleotide reductase à severe immunodeficiency disease (T and B lymphocytes do not develop properly). EXCESS URIC ACID CAUSES GOUT q Gout is a disease of the joints caused by high levels of uric acid in the blood and tissues (à abnormal deposition of urate crystals in the joints and kidneys à inflammation, pain and arthritis). q Occurs predominantly in males. q Cause is unknown but involves underexcretion of urate. Allopurinol, an inhibitor of xanthine oxidase. Hypoxanthine is the normal substrate of xanthine oxidase. Only a slight alteration in the structure of hypoxanthine (light red) yields the medically effective enzyme inhibitor allopurinol. At the active site, allopurinol is converted to oxypurinol, a strong competitive inhibitor that remains tightly bound to the reduced form of the enzyme. q Effectively treated by a combination of diet (food rich in nucleotides and nucleic acids such as liver are withheld) and drug (allopurinol). Allopurinol inhibits xanthine oxidase, the enzyme that catalyzes the conversion of purines to uric acid. CATABOLISM OF PYRIMIDINES PRODUCES UREA AND INTERMEDIATES OF THE CAC Pyrimidine ring is opened and degraded to highly soluble products Leads to b-alanine (from CMP and UMP degradation) and b-aminoisobutyrate (from TMP degradation) Production of NH4+ (then urea) and CO2 Can produce intermediates of CAC – Example: Thymine is degraded to succinyl-CoA and uracil to acetyl CoA CATABOLISM OF PYRIMIDINES PRODUCES UREA AND INTERMEDIATES OF THE CAC Leads to NH4+ then urea Can produce intermediates of CAC –Example: Thymine is degraded to succinyl-CoA Succinyl CoA