23a-Biochemistry-Lecture23a.pdf

Principles of Biochemistry SPRING 2024 Professor: Moncef LADJIMI [email protected] Office: C-169 As faculty of Weill Cornell Medical College in Qatar we are committed to providing transparency for any and all external relationships prior to giving an academic presentation. I, Moncef LADJ...

Principles of Biochemistry SPRING 2024 Professor: Moncef LADJIMI [email protected] Office: C-169 As faculty of Weill Cornell Medical College in Qatar we are committed to providing transparency for any and all external relationships prior to giving an academic presentation. I, Moncef LADJIMI DO NOT have a financial interest in commercial products or services. Lecture 23a Lipid Metabolism: Fatty Acid Oxidation Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 17: p. 601-621 FATTY ACID OXIDATION Key topics: – How fats are digested in animals – How fats are mobilized and transported in tissues – How fats are oxidized – How “ketone bodies” are produced OXIDATION OF FATTY ACIDS IS A MAJOR ENERGY SOURCE IN MANY ORGANISMS About one-third of our energy needs comes from dietary triacylglycerols. However, many hibernating animals, such as grizzly bears, rely almost exclusively on fats as their source of energy About 80% of energy needs of mammalian heart and liver are met by oxidation of fatty acids Fuel reserves in a typical ~70 Kg man 100,000 kcal in triacylglycerols (~15 kg) 25,000 kcal in protein (~5 kg) 600 kcal in glycogen (~0.2 kg) 40 kcal in glucose (~0.02 kg) FATS PROVIDE EFFICIENT FUEL STORAGE The advantage of fats over polysaccharides: – Fatty acids carry more energy per carbon because they are more reduced – Fatty acids carry less water along because they are nonpolar Glucose and glycogen are for short-term energy needs, quick delivery Fats are for long-term (months) energy needs, good storage, slow delivery LIPID CATABOLISM: OXIDATION OF FATTY ACIDS § Oxydation of long chain fatty acids (LCFA) to acetyl-CoA: energy yielding pathway (80% of the energetic needs) § Electrons removed from LCFA during oxydation pass through the respitory chain driving ATP synthesis § The acetyl-CoA produced may be completely oxidized to CO2 in the citric acid cycle, resulting in further energy conservation § when glucose is not available, acetyl-CoA may be converted to ketone bodies in liver, fuel exported to the brain and other tissues OUTLINE OF FATTY ACIDS OXIDATION § sources: dietary and stored fatty acis § getting fatty acids into cells § carnitine shuttle to take fatty acids into the mitochondrial matrix for oxidation § reactions of b-oxidation: 4-step cycle § oxidation of saturated vs unsaturated fatty acids § production of ketone bodies in liver 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. 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. 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 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 STAGES OF FATTY ACID OXIDATION How is fat oxidized to provide a source of metabolic energy (ATP)? Stage 1 : b-oxidation; consists of oxidative conversion of two-carbon units into acetyl-CoA with concomitant generation of NADH and FADH2 – involves oxidation of β carbon to thioester of fatty acyl-CoA Stage 2: TCA cycle involves oxidation of acetyl-CoA into CO2 via citric acid cycle with concomitant generation NADH and FADH2 Stage 3: Oxidative phosphorylation generates ATP from NADH and FADH2 via the respiratory chain Stage 1: A long-chain fatty acid is oxidized to yield acetyl residues in the form of acetyl-CoA. This process is called β oxidation. Stage 2: The acetyl groups are oxidized to CO2 via the citric acid cycle. Stage 3: Electrons derived from the oxidations of stages 1 and 2 pass to O2 via the mitochondrial respiratory chain, providing the energy for ATP synthesis by oxidative phosphorylation. 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 STRATEGY OF b-OXIDATION § The C-C bond between methylene groups in fatty acids is quite stable. § The first 3 reactions of b-oxidation create a much less stable C-C bond in which C2 (Ca) is bonded to two carbonyl carbons (b-ketoacyl-CoA intermediate). § The ketone function on the b carbon (C3) makes it a good target for nucleophilic attack by the SH group of CoA catalyzed by Thiolase b a § Thiolase splits of the 2-Carbons fragment in the 4th reaction. SIMILAR MECHANISMS INTRODUCE CARBONYLS IN OTHER METABOLIC PATHWAYS Fatty-acyl CoA Succinate Isoleucine A conserved reaction sequence to introduce a carbonyl function on the carbon β to a carboxyl group. -The β-oxidation pathway for fatty acyl–CoAs to b-ketoacyl-CoA -The pathway from succinate to oxaloacetate in the citric acid cycle, b-keto-acyl CoA Oxaloacetate -the pathway by which the deaminated carbon skeletons a-methyl acetoacetyl CoA from isoleucine, leucine, and valine are oxidized as fuels, use the same reaction sequence. 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 TRIFUNCTIONAL PROTEIN CATALYZES THE THREE LAST STEPS FOR FATTY ACIDS LONGER THAN 12 CARBONS Trifunctional protein is a Hetero-octamer – Four a subunits enoyl-CoA hydratase activity b-hydroxyacyl-CoA dehydrogenase activity Responsible for binding to membrane – Four b subunits long-chain thiolase activity May allow substrate channeling Associated with inner-mitochondrial membrane Processes fatty acid chains with 12 or more carbons Shorter chains are processed by soluble enzymes in the matrix 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 UNSATURATED FATTY ACIDS Naturally occurring Unsaturated Fatty acids contain cis double bonds – Are NOT a substrate for enoyl-CoA hydratase Two additional enzymes are required – An isomerase: converts cis double bonds starting at carbon 3 to trans double bonds – A reductase: reduces cis double bonds not at carbon 3 u Monounsaturated fatty acids require only the isomerase u Polyunsaturated fatty acids require both the isomerase and the reductase OXYDATION OF MONO-UNSATURATED FATTY ACIDS REQUIRES ONE ADDITIONAL STEP Example: Oxidation of oleic acid (18:1(∆9)): mono-unsaturated § Recall: Enoyl-CoA hydratase acts on trans double bonds (thus, the double bond requires isomerization) § An auxiliary enzyme (∆3,∆2enoyl-CoA isomerase) is needed § Energy yield is less since unsaturated fatty acids are already partially oxidized: at the level of the double bonds, step 1 is bypassed à no FADH2 formation (-1,5 ATP) Oxidation of a monounsaturated fatty acid. Oxidation of oleic acid, as oleoyl-CoA (Δ9) requires an additional enzyme, enoyl-CoA isomerase, to reposition the double bond, converting the cis isomer to a trans isomer, a normal intermediate in β oxidation. OXYDATION OF POLY-UNSATURATED FATTY ACIDS REQUIRES TWO ADDITIONAL STEPS Example: Oxidation of linoleic acid (18:1(∆9,12)): polyunsaturated § Enoyl-CoA hydratase acts on trans double bonds (the first double bond requires isomerization while the second requires reduction/isomerization). Therefore: § A first auxiliary enzyme (∆3,∆2-enoyl-CoA isomerase) is needed (the first double bond requires isomerization; see monounsaturated Fatty acids) § A second auxiliary enzyme is needed (the second double bond requires reduction/isomerisation) to allow the complete oxidation of a polyunsaturated fatty acid, e.g., linoleic acid (18:2(∆9,∆12)) § energy yield ~less since unsaturated fatty acids are already partially oxidized: at the level of the double bonds, step 1 is bypassed à no FADH2 formation (-1,5 ATP) Oxidation of a polyunsaturated fatty acid Oxidation requires a second auxiliary enzyme in addition to enoylCoA isomerase: the NADPH-dependent 2,4-dienoyl-CoA reductase. The combined action of these two enzymes converts a trans-Δ2,cisΔ4-dienoyl-CoA intermediate to the trans-Δ2-enoyl-CoA substrate necessary for β oxidation. OXIDATION OF ODD-NUMBERED FATTY ACIDS Most dietary fatty acids are even-numbered Many plants and some marine organisms synthesize odd-numbered fatty acids Oxydation of odd-numbered fatty acids from food by b-oxidation (2-carbons at a time) leads to the formation of the 3-carbons Propionyl-CoA Propionyl-Co is then converted to succinyl-CoA and enters the citric acid cycle. Bacterial metabolism in the rumen of ruminants also produces propionyl-CoA 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 SUMMARY Fats are an important energy source in animals Two-carbon units in fatty acids are oxidized in a four-step b-oxidation process into acetyl-CoA In the process, a lot of NADH and FADH2 forms; these can yield a lot of ATP in the electrontransport chain and oxphos Acetyl-CoA formed in the liver can be either oxidized via the citric acid cycle or converted to ketone bodies that serve as fuels for other tissues Remember to prepare for next lecture: Lehninger’s Biochemistry (8th ed), §chapter 21: p. 744-771