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Lipid Metabolism III; amino acid metabolism I

Lipid Metabolism III; amino acid metabolism I. Andy Howard Biochemistry Lectures, Fall 2010 15 November 2010. Lipid catabolism; amino acid metabolism. We’ll talk about control of lipid catabolism and some special topics

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Lipid Metabolism III; amino acid metabolism I

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  1. Lipid Metabolism III; amino acid metabolism I Andy HowardBiochemistry Lectures, Fall 201015 November 2010 Biochem: lipid 3, aa 1

  2. Lipid catabolism; amino acid metabolism • We’ll talk about control of lipid catabolism and some special topics • Then we’ll begin an in-depth conversation about amino acid synthesis and degradation Biochem: lipid 3, aa 1

  3. Lipids, concluded Odd-chain and unsaturated FAs Regulation by hormones Absorption and mobilization Lipoproteins Ketone bodies Amino acid synthesis Nitrogen sources Nitrogenase Essential aa’s Transaminations Non-essential amino acids Branched-chain Aromatics What we’ll cover Biochem: lipid 3, aa 1

  4. Methylmalonyl CoA Odd-chain fatty acids • Rarer than even-chain but they do exist • Broken down as with even-chains but with propionyl CoA as end-product • Condenses with bicarbonate to form D-methylmalonyl CoA • Racemized to L-methylmalonyl CoA • Mutated to succinoyl CoA via an adenosylcobalamin-dependent reaction • This can actually be a source of sugars! Biochem: lipid 3, aa 1

  5. Catabolism of cis-unsaturated 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 Biochem: lipid 3, aa 1

  6. Regulation I epinephrine • Key hormones:insulin, glucagon, epinephrine • Under low-glucose conditions: • glucagon and epinephrine circulate at high concentrations • -oxidation encouraged • Glucose not needed for fuel so it’s conserved • High glucose conditions: • insulin, glucagon & epinephrine , • FA synthesis dominates • Glucose used as fuel for making fatty acids Biochem: lipid 3, aa 1

  7. 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 Carnitine palmitoyl -transferasePDB 2RCUEC 2.3.1.21 1.8Å148 kDa dimer Monomer shown Biochem: lipid 3, aa 1

  8. 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 Biochem: lipid 3, aa 1

  9. Fates of triacylglycerols • What happens next depends on needs: • Triacylglycerols hydrolyzed to FAs and monoacylglycerols, and sometimes further • High [insulin] inhibits hydrolysis Biochem: lipid 3, aa 1

  10. GlucagonPDB 1GCN, 3Å3.3kDa monomer Glycerol and free fatty acids • 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 Biochem: lipid 3, aa 1

  11. 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 From Y.Shi & P.Burn (2004) Nature Rev. Drug Discov.3: 695. Biochem: lipid 3, aa 1

  12. Lipase and colipase • Pancreatic lipase secreted into small intestine degrades triacylglycerols (in fat particles) at C-1&3 • Colipase: 10.4 kDa protein that helps bind the lipase to its substrates Lipase-colipase complexPDB 1LPB10.4 kDa (colipase)+ 50kDa (lipase)heterodimerEC 3.1.1.3, 2.46Å Pig / human Biochem: lipid 3, aa 1

  13. 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 Biochem: lipid 3, aa 1

  14. Dietary phospholipids • Phospholipase A2 in intestine hydrolyzes ester bond at C2; the resulting lysophosphoglycerides getre-esterified in the intestine • High [lysophosphoglyceride] disrupts membranes: that’s how snake venoms work on erythrocytes PDB 1G4I13.5 kDa monomerBovine pancreasEC 3.1.1.40.97Å Biochem: lipid 3, aa 1

  15. 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 cholesteryl esters Cholesterol esterasePDB 2BCE64 kDa monomerBovineEC 3.1.1.16 1.6Å Biochem: lipid 3, aa 1

  16. Core Lipoproteins • Spherical vehicles fortransport of fats • Several sizes • Biggest, least dense:chylomicrons • Others are smaller,more dense Cartoon courtesyU. WisconsinStevens Point Biochem: lipid 3, aa 1

  17. Chylomicrons • Largest, least dense oflipoproteins • found in bloodonly after a meal • Deliver triacylglycerol &cholesterol to muscle and adipose tissue • Remaining cholesterol-rich particles deliver cholesterol to liver • Contains Apolipoprotein E -binds to specific receptor in liver cells Biochem: lipid 3, aa 1

  18. Types of lipoproteins(cf. table 16.1 & fig. 16.30) Type Chylo- VLDLs IDLs LDLs HDLs microns MW*10-6 >400 10-80 5-10 2.3 .18-.36 , g cm-3 <0.95 <1.006 <1.019 <1.063 <1.21 Composition (%) Protein 2 10 18 25 33 Triacylglycerol 85 50 31 10 8 Cholesterol 4 22 29 45 30 Phospholipid 9 18 22 20 29 Biochem: lipid 3, aa 1

  19. % protein and density Biochem: lipid 3, aa 1

  20. Protein components • Structural amphipathic 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 Kringle domain of ApoA1 PDB 3KIV 8.7 kDa monomer Human 1.8Å Biochem: lipid 3, aa 1

  21. Low-density lipoproteins • LDLs deliver cholesterol to peripheral tissues via cell-surface binding • High intracellular [cholesterol] inhibits synthesis of HMGCoA reductase and the LDL 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” Biochem: lipid 3, aa 1

  22. 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” Biochem: lipid 3, aa 1

  23. Serum albumin • Free fatty acids carried by this protein • 7 binding sites for FAs • Human Serum Albumin also binds many hydrophobic drugs HSA + 7 palmitatesPDB 1E7H68 kDa monomer, 2.43Å Biochem: lipid 3, aa 1

  24. Ketone bodies acetone aceto-acetate • 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 -hydroxy-butyrate Biochem: lipid 3, aa 1

  25. 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 HMGCoA lyasePDB 2CW6EC 4.1.3.4, 2.1Å197 kDa hexamer human Biochem: lipid 3, aa 1

  26. 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 Succinyl CoA transferasePDB 1OOY212 kDaEC 2.8.3.51.7Åtetramer; dimer shownpig heart Biochem: lipid 3, aa 1

  27. 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 Biochem: lipid 3, aa 1

  28. 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 Biochem: lipid 3, aa 1

  29. 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 Biochem: lipid 3, aa 1

  30. Structural features of nitrogenase • Multi-component complex • Mo-Fe active site in actual N2-fixing component • Probably proceeds via diimine and hydrazine: • NN + 2e- + 2H+ H-N=N-H • H-N=N-H + 2e- + 2H+ H2N-NH2 • H2N-NH2 + 2e- + 2H+ 2 NH3 • 2e- + 2H+ H2  Nitrogenase Mo-Fe + Fe proteinsPDB 1G20350 kDa hetero-octamerEC 1.18.6.1, 2.2ÅAzotobacter Biochem: lipid 3, aa 1

  31. 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 Nitrate reductasePDB 2BO0111 kDa trimermonomer shownAlcaligines Biochem: lipid 3, aa 1

  32. 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 Biochem: lipid 3, aa 1

  33. AA moles ATP essen- tial? Asp 21 no Asn 22-24 no Lys 50-51 yes Met 44 yes Thr 31 yes Ala 20 no Val 39 yes Leu 47 yes Ile 55 yes Glu 30 no Gln 31 no AA moles ATP essen- tial? Arg 44 no Pro 39 no Ser 18 no Gly 12 no Cys 19 no Phe 65 yes Tyr 62 no* Trp 78 yes His 42 yes The human list (~ box 17.3) Biochem: lipid 3, aa 1

  34. Transaminations • General process of interconverting -amino acids and -ketoacids • Primary way that N gets incorporated into non-N-containing structures Biochem: lipid 3, aa 1

  35. Reaction dynamics • All (?) transaminations involve PLP as a cofactor: see mechanism, fig. 7.18 • These are actually oxidation-reduction 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 Aspartateaminotransferase PDB 2Q7W87 kDa dimer; Monomer shownEC 2.6.1.1, 1.4Å E.coli Biochem: lipid 3, aa 1

  36. Examples of transaminases Reactants Products Trans- aminase Keto acid amino acid keto acid amino acid Pyruvate glutamate -k-glutarate alanine pyruvate Pyruvate aspartate oxaloacetate alanine pyruvate Oxaloacetate glutamate -k-glutarate aspartate aspartate 3-phosphono- glutamate -k-glutarate phosphoserine phospo- hydroxypyruvate serine 3-OH-phenyl- glutamate -k-glutarate tyrosine tyrosinepyruvate Biochem: lipid 3, aa 1

  37. Catabolic or anabolic? • From the point of view of available pools of amino acids, these are amphibolic: • They involve synthesis of one amino acid at the expense of another Biochem: lipid 3, aa 1

  38. Some are complex and energy-requiring Can be logically divided according to chemical properties of the target amino acids: Small Branched-chain aliphatic Neutral polar Acidic Basic Aromatic Sulfur-containing Biosynthetic pathways to specific amino acids Biochem: lipid 3, aa 1

  39. Which amino acids in which categories? Category Amino acids • Small gly, ala, ?pro? • Branched-chain val, leu, ile aliphatic • Neutral polar asn, gln, ser, thr • Acidic asp, glu • Basic lys, arg • Aromatic phe, tyr, trp, his • Sulfur-containing cys, met Biochem: lipid 3, aa 1

  40. 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 Glu dehydrogenase PDB 1BGV296 kDa hexamermonomer shown EC 1.4.1.2, 1.9ÅClostridium Biochem: lipid 3, aa 1

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