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Lipid Catabolism, concluded; amino acid metabolism Andy Howard Introductory Biochemistry 22 April 2008 What we’ll cover Lipids, concluded Similarities & differences Trafficking Bookkeeping Special Cases Locations for synthesis Regulation by hormones Absorption and mobilization Lipoproteins Ketone bodies Lipid & Amino Acid Metabolism Amino acid metabolism Nitrogen sources Nitrogenase Essential aa’s Transaminations General categories Specific pathways p. 2 of 44 22 April 2008 Similarities and differences … between synthesis and oxidation of fatty acids: Acetyl CoA Malonyl ACP fatty acid Fatty acid FA CoA acetyl CoA Text gives clear exposition: read it! This could easily result in a final exam question! Lipid & Amino Acid Metabolism p. 3 of 44 22 April 2008 Moving acyl CoA into the mitochondrial matrix Carnitine shuttle moves them: Fatty acyl CoA gets into the intermembrane space Carnitine acyltransferase II acylates carnitine Acylcarnitine crosses inner membrane Similar enzyme inside gets acyl CoA back Lipid & Amino Acid Metabolism p. 4 of 44 Carnitine: 3-hydroxy-4trimethylammoniobutanoate 22 April 2008 Bookkeeping Remember: mitochondrial or bacterial FA oxidation yields one NADH and one QH2 per pair of carbons, e.g. for stearate (C18): Stearoyl CoA + 8HS-CoA + 8Q + 8NAD+ 9 acetyl CoA + 8QH2 + 8NADH +8H+ That adds up to 12+20+90-2 = 120 ATP! Lipid & Amino Acid Metabolism p. 5 of 44 22 April 2008 Comparisons with glucose Glucose is 6 carbons, not 18, so the 120 ATPs vs. 32 comes out more like 120 vs.96 on a percarbon basis… But per gram: 1 g * 32 mol ATP (mol glucose) / 180 g/mol = 32/180 = 0.178 moles ATP per gram glucose 1 g * 120 mol ATP /(mol stearate) / 288 g/mol) = 120/288 = 0.417 moles ATP per gram stearate Hydration matters too! Lipid & Amino Acid Metabolism p. 6 of 44 22 April 2008 Efficiency of fatty acyl synthesis 8 acetyl CoA 8 malonyl ACP = 8 ATP 8 syntheses * 5 NADPH/synthesis = 40 9 Acetyl CoA * 17 ATP/AcCoA = 153 Total: 201 ATP So efficiency = 120/201 = 0.597 That’s a typical efficiency defined as (energy derived from oxidation) / (energy required for synthesis) Lipid & Amino Acid Metabolism p. 7 of 44 22 April 2008 Odd-chain fatty acids Methylmalonyl CoA Rarer than even-chain but they do exist Broken down as with even-chains but with priopionyl CoA as end-product Condenses with bicarbonate to form Dmethylmalonyl CoA Racemized to L-methylmalonyl CoA Mutated to succinoyl CoA via an adenosylcobalamin-dependent reaction This can actually be a source of sugars! Lipid & Amino Acid Metabolism p. 8 of 44 22 April 2008 Catabolism of cisunsaturated fatty acids Normal beta-oxidation until we encounter a double bond Double bond moves from cis-3,4 to trans-2,3 via 3,2-enoyl-CoA isomerase reaction Further beta oxidation proceeds until we encounter the next double bond; Cis double bonds at even positions get modified by 2,4-dienoyl-CoA reductase from trans,cis-2,4 to trans-3 3,2-enoyl-CoA isomerase moves trans-3 to trans-2 and then we can -oxidize again Lipid & Amino Acid Metabolism p. 9 of 44 22 April 2008 Regulation I Key hormones: insulin, glucagon, ephinephrine Under low-glucose conditions: glucagon and ephinephrine circulate at high concentrations -oxidation encouraged Glucose not needed for fuel so it’s conserved High glucose conditions: insulin, glucagon & ephinephrine , FA synthesis dominates Glucose used as fuel for making fatty acids Lipid & Amino Acid Metabolism p. 10 of 44 22 April 2008 Regulation II Main regulatory enzyme: acetyl-CoA carboxylase High insulin levels after meal stimulates formation of malonyl CoA Product allosterically inhibits carnitine acyltransferase so FAs stay in cytosol Lipid & Amino Acid Metabolism p. 11 of 44 Carnitine palmitoyl transferase PDB 2RCU 144 kDa dimer Monomer shown 22 April 2008 Mobilization of fatty acids Triacylglycerols transported through circulatory system in lipoprotein masses (cholesterol + various MW proteins forming shell around lipid) Lipoproteins hydrolyzed via lipoprotein lipase extracellularly Fatty acids & glycerol released extracellularly, FAs re-esterified What happens next depends on needs: Triacylglycerols hydrolyzed to FAs and monoacylglycerols, and sometimes further High [insulin] inhibits hydrolysis Lipid & Amino Acid Metabolism p. 12 of 44 22 April 2008 Glycerol and free fatty acids Glucagon PDB 1GCN 3.3kDa monomer Some of them diffuse through the adipocyte plasma membrane & enter blood Glycerol metabolized in liver to (…) glucose FAs travel bound to serum albumin to heart, skeletal muscle, & liver— energy source esp. in fasting Glucagon means inhibition of acetyl CoA carboxylase, so less malonyl CoA made Meanwhile: high [acetyl CoA],[NADH] means inhibition of pyruvate dehydrogenase Lipid & Amino Acid Metabolism p. 13 of 44 22 April 2008 Absorption of lipids from food Majority of dietary lipids are triacylglyerols; Smaller amounts of phospholipids & cholesterol Suspended fat particles are coated with bile salts, amphipathic cholesterol derivatives Pancreatic lipase secreted into small intestine degrades triglycerides (in fat particles) at C-1&3 Colipase helps bind the lipase to its substrates Lipid & Amino Acid Metabolism p. 14 of 44 Lipase-colipase complex PDB 1LPB 10.4 kDa+ 50kDa heterodimer 22 April 2008 What bile salts do Taurocholate, a bile salt Bile-salt micelles travel to intestinal wall Monoacylglycerols & free FAs are absorbed and bile salts are released Bile salts recirculate rapidly When fully formed triglycerides are made, those travel via chylomicrons for transport to other tissues Lipid & Amino Acid Metabolism p. 15 of 44 22 April 2008 Dietary phospholipids Phospholipase A2 in intestine hydrolyzes ester bond at C2; the resulting lysophosphoglycerides get re-esterified in the intestine High [lysophosphoglyceride] disrupts membranes: that’s how snake venoms work on erythrocytes Lipid & Amino Acid Metabolism p. 16 of 44 PDB 1G4I 13.5 kDa monomer Bovine pancreas 22 April 2008 Dietary cholesterol Cholesterol esters are hydrolyzed in the lumen of the intestine Free cholesterol is solubilized by bile-salts for absorption Free cholesterol often esterified in the intestine to form choesteryl esters Lipid & Amino Acid Metabolism p. 17 of 44 Cholesterol esterase PDB 2BCE 64 kDa monomer Bovine 22 April 2008 Lipoproteins Core Spherical vehicles for transport of fats Several sizes Biggest, least dense: chylomicrons found in blood only after a meal Cartoon courtesy Deliver triacylglycerol & cholesterol to U. Wisconsin muscle and adipose tissue Stevens Point Remaining cholesterol-rich particles deliver cholesterol to liver Contains Apolipoprotein E - binds to specific receptor in liver cells Others are smaller, Lipid & Amino Acid Metabolism more dense p. 18 of 44 22 April 2008 Types of lipoproteins (cf. table 16.1 & fig. 16.30) Type Chylo- VLDLs IDLs LDLs microns MW*10-6 >400 10-80 5-10 2.3 , g cm-3 <0.95 <1.006 <1.019 <1.063 Composition (%) Protein 2 10 18 25 Triacylglycerol 85 50 31 10 Cholesterol 4 22 29 45 Phospholipid 9 18 22 20 Lipid & Amino Acid Metabolism p. 19 of 44 HDLs .18-.36 <1.21 33 8 30 29 22 April 2008 % protein and density 1.12 Density depends on % protein 1.1 HDLs 1.08 Density, g cm-3 1.06 1.04 LDLs 1.02 IDLs 1 0.98 VLDLs 0.96 0.94 0.92 Chylomicrons % protein 0.9 0 5 10 Lipid & Amino Acid Metabolism 15 20 p. 20 of 44 25 30 35 22 April 2008 Protein components Structural amiphathic crust proteins: ApoB-100 (513 kDa) bound to outer layer of VLDLs, IDLs, LDLs. ApoB-48 (241 kDa): N-terminal end of ApoB-100, found in chylomicrons Smaller, less strongly bound proteins Some are responsible for specific binding to receptors in cells Lipid & Amino Acid Metabolism p. 21 of 44 Kringle domain of ApoA1 PDB 3KIV 8.7 kDa monomer Human 22 April 2008 Low-density lipoproteins LDLs deliver cholesterol to peripheral tissues via cell-surface binding High intracellular [cholesterol] inhibits synthesis of HMGCoA reductase and the receptor People without LDL receptor: cholesterol accumulates in the blood and gets deposited in skin and arteries This risk leads to the description of LDLs as “bad cholesterol” Lipid & Amino Acid Metabolism p. 22 of 44 22 April 2008 High-density lipoproteins Take cholesterol out of plasma and return it to the liver Binds to receptor SR-B1 and transfer cholesterol & cholesterol esters back to liver cells Lipid-depleted HDLs return to plasma Because these tend to deplete cholesterol from the bloodstream, they become known as “good cholesterol” Lipid & Amino Acid Metabolism p. 23 of 44 22 April 2008 Serum albumin Free fatty acids carried by this protein 7 binding sites for Fas HSA also binds many hydrophobic drugs Lipid & Amino Acid Metabolism HSA + 7 palmitates PDB 1E7H 64 kDa monomer p. 24 of 44 22 April 2008 acetone Ketone bodies Three compounds produced as stored-fuel molecules -hydroxybutyrate & acetoacetate are fuel Serve as water-soluble lipids— readily transported in plasma Important in brain, skeletal muscle, intestine during starvation Lipid & Amino Acid Metabolism p. 25 of 44 acetoacetate hydroxybutyrate 22 April 2008 Synthesis of ketone bodies Starts out like steroids: 2 acetyl CoA acetoacetyl CoA HMG CoA Then HMG CoA lyase converts HMG CoA to acetoacetate and acetyl CoA Acetoacetate can be reduced via NADH to -hydroxybutyrate Acetoacetate can also be nonezymatically decarboxylated to acetone Lipid & Amino Acid Metabolism p. 26 of 44 HMGCoA lyase PDB 2CW6 EC 4.1.3.4 197 kDa hexamer human 22 April 2008 Oxidation of ketone bodies -hydroxybutyrate oxidized back to acetoacetate in a separate version of the liver enzyme that made it acetoacetate converted to acetoacetyl CoA in mitochondria in nonhepatic tissues via succinyl-CoA transferase Thiolase converts acetoacetyl CoA into two molecules of acetyl CoA Lipid & Amino Acid Metabolism p. 27 of 44 Succinyl CoA transferase PDB 1OOY 212 kDa tetramer dimer shown pig heart 22 April 2008 Amino acid metabolism As Horton says, this is a difficult subject to cover Hundreds of reactions, dozens of reaction pathways Some common threads and generalizations We’ll focus on the latter Lipid & Amino Acid Metabolism p. 28 of 44 22 April 2008 The nitrogen pool (fig. 17.1) Nitrogen fixation from air (N2 NH3) doesn’t produce a large percentage of circulating biological nitrogen but it’s the ultimate source of most of it Other entries in pool: nitrate (NO3 -), nitrite (NO2-) Most of this difficult biochemistry is bacterial Lipid & Amino Acid Metabolism p. 29 of 44 22 April 2008 Nitrogenase Enzyme found in Rhizobium, a bacterium that colonizes & lives symbiotically in the root nodules of legumes and a few other plants Also in free-living microorganisms like Azotobacter Energetically expensive but irreversible path to reduction of dinitrogen to ammonia: N2 + 8H+ + 8e- + 16 ATP 2NH3 + H2 + 16ADP + 16Pi Lipid & Amino Acid Metabolism p. 30 of 44 22 April 2008 Structural features of nitrogenase Multi-component complex Mo-Fe active site in actual N2fixing component Nitrogenase Probably proceeds via diimine and Mo-Fe + Fe proteins hydrazine: N=N + + H-N=N-H H-N=N-H + 2e- + 2H+ H2N-NH2 H2N-NH2 + 2e- + 2H+ 2 NH3 2e- 2H+ Lipid & Amino Acid Metabolism p. 31 of 44 PDB 1G20 350 kDa heterooctamer Azotobacter 22 April 2008 Ammonia, nitrate, nitrite Ammonia comes from decayed organisms and is oxidized in soil bacteria to nitrate (nitrification) Nitrate reductase and nitrite reductase found in plants and microorganisms: NO3- + 2e- + 2H+ NO2- + H2O NO2- + 6e- + 7H+ NH3 + 2 H2O Lipid & Amino Acid Metabolism p. 32 of 44 Nitrate reductase PDB 2BO0 111 kDa trimer monomer shown Alcaligines 22 April 2008 Essential and non-essential amino acids An amino acid is defined as essential if it must be obtained within the diet In general the essential amino acids are the ones that have complicated and highly ATP-dependent biosynthetic pathways Of course, it depends on the organism Lipid & Amino Acid Metabolism p. 33 of 44 22 April 2008 The human list (cf. box 17.3) AA moles ATP Asp Asn Lys Met Thr Ala Val Leu Ile 21 22-24 50-51 44 31 20 39 47 55 essential? no no yes yes yes no yes yes yes Glu Gln 30 31 no no Lipid & Amino Acid Metabolism AA moles ATP Arg Pro Ser Gly Cys Phe Tyr Trp His 44 39 18 12 19 65 62 78 42 p. 34 of 44 essential? no no no no no yes no* yes yes 22 April 2008 Transaminations General process of interconverting amino acids and -ketoacids Primary way that N gets incorporated into non-N-containing structures Lipid & Amino Acid Metabolism p. 35 of 44 22 April 2008 Reaction dynamics All (?) transaminations involve PLP as a cofactor: see mechanism, fig. 7.18 These are actually oxidationreduction reactions, since we’re swapping an amine (carbon oxidation state +2) for a carbonyl (carbon oxidation state 0) But there is no external oxidizing agent Lipid & Amino Acid Metabolism p. 36 of 44 Aspartate aminotransferase PDB 2Q7W 87 kDa dimer; Monomer shown E.coli 22 April 2008 Examples of transaminases Reactants Products Keto acid amino acid Pyruvate glutamate Pyruvate aspartate Oxaloacetate glutamate 3-P-OH-pyr glutamate 3-OH-phenyl- glutamate pyruvate keto acid -k-glutarate oxaloacetate -k-glutarate -k-glutarate a-k-glutarate Lipid & Amino Acid Metabolism Transaminase amino acid alanine pyruvate alanine pyruvate aspartate aspartate P-ser phosphoserine tyrosine tyrosine p. 37 of 44 22 April 2008 Catabolic or anabolic? From the point of view of available pools of amino acids, these are ambiphibolic: They involve synthesis of one amino acid at the expense of another Lipid & Amino Acid Metabolism p. 38 of 44 22 April 2008 Biosynthetic pathways to specific amino acids Some are complex and energy-requiring Can be logically divided according to chemical properties of the target amino acids: Lipid & Amino Acid Metabolism Small Branched-chain aliphatic Neutral polar Acidic Basic Aromatic Sulfur-containing p. 39 of 44 22 April 2008 Glutamate Glutamate is a critical metabolite because so many of the transaminations start with it as the amine donor It is produced in E.coli, etc. via glutamate dehydrogenase using ammonium ion as nitrogen donor: -ketoglutarate + NH4+ + NAD(P)H + H+ NAD(P)+ + H2O + glutamate Lipid & Amino Acid Metabolism p. 40 of 44 1BGV 296 kDa hexamer monomer shown Clostridium 22 April 2008 Glutamine Glutamate can be aminated with expenditure of ATP to form glutamine: glutamate + NH4+ + ATP glutamine + ADP + Pi Note that glutamine synthetase is a ligase: the ATP is an energy-provider, not a phosphate donor Lipid & Amino Acid Metabolism p. 41 of 44 Glutamine synthetase PDB 2OJW 211 kDa pentamer human 22 April 2008 Aspartate and asparagine Asp is simple: transamination of oxaloacetate Asn is straightforward too asparagine synthetase moves the amine from gln to asp, leaving glu (another lyase) Gln + asp + ATP AMP + PPi + glu + asn Lipid & Amino Acid Metabolism p. 42 of 44 Asparagine synthetase B PDB 1CT9 243 kDa tetramer E.coli 22 April 2008 Simple: ala, gly, ser Alanine by transamination from pyruvate Glycine from serine by SHMT (q.v.) Serine from 3-phosphoglycerate: Phosphoserine phosphatase 3-phosphoglycerate + NAD PDB 1NNL NADH + H+ + 349 kDa dimer phosphohydroxypyruvate human 3-phosphohydroxypyruvate + glutamate 3-phosphoserine + -ketoglutarate 3-phosphoserine + H2O serine + Pi Lipid & Amino Acid Metabolism p. 43 of 44 22 April 2008 Serine hydroxymethyltransferase Serine + tetrahydrofolate PDB 2DKJ H2O + glycine + 5,10-methylene- 90 kDa dimer Thermus tetrahydrofolate thermophilus This can be viewed as a source of methylene units for other biosyntheses PLP-dependent reaction Lipid & Amino Acid Metabolism p. 44 of 44 22 April 2008