Lipid Metabolism, Fatty Acid Oxidation

Lipases are a family of proteins that are specific for were they hydrolyse triaclygycerides molecule and what tissues cells they are expressed.

Mechanism of interfacial activation of triacylglycerol lipase (pancreatic lipase) in complex with colipase. This enzyme cleaves at the 1 & 3 position forming 1,2 diacylgycerols & 2-acylglycerol. Colipase is secreted by the pancreas as procolipase, it is activated by trypsin. Colipase is a small peptide with 5 conserved disulfide bonds.

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Catalytic triad & colipase •  Catalytic activity increases with the formation of a complex with

coplipase at the C-terminal Colipase stablizes the open configuration by binding to the C-terminal and H bonding to the open lid.

•  The active site is in the N-terminal and has a catalytic triad that resembles serine protease

•  Without lipid micelles presence, a 25 aa residue lid covers active site

•  When in the presence of a micelle the β5 loop changes conformation to expose the oxyanion hole of active site.

•  Phospholipids are degraded by phospholipase A2. •  Phospholipase A2 contains a hydrophobic channel that allows direct

access of the substrate to the active site. It has a catalytic dyad that utilizes a bound H2O molecule in the presence of Ca+2.

•  The Ca+2 stabilizes the oxyanion transition.

Substrate binding to phospholipase A2. The model of phospholipase A2 in complex with a micelle of lysophosphatidylethanolamine. Requires Ca+2 For activity. Due to PLA2 role in inflammation it is also regulated by phosphorylation, on a Ser residue by MAPK.

•  The catalytic mechanism of pancreatic sPLA2 is initiated by a His-48 & Asp-99 Ca+2 complex within the active site. The Ca+2 polarizes the SN-2 carbonyl oxygen while also coordinating with a catalytic water molecule. His-48 improves the nucleophilicity of the catalytic water via a bridging second water molecule. It has been suggested that two water molecules are necessary to traverse the distance between the catalytic histidine and the ester. The basicity of His-48 is thought to be enhanced through H bonding with Asp-99. The rate limiting state is characterized as the degradation of the tetrahedral intermediate composed of a Ca+2 coordinated oxyanion. PLA2 can also be characterized as having a channel featuring a hydrophobic wall in which hydrophobic amino acid residues such as Phe, Leu, and Tyr serve to bind the substrate. Another component of PLA2 is the seven disulfide bridges that are influential in regulation and stable protein folding.

•  Hormone-sensitive lipase (HSL) also previously known as cholesteryl ester hydrolase (CEH) is an enzyme that, in humans, is encoded by the LIPE gene.

•  HSL is an intracellular neutral lipase that is capable of hydrolyzing a variety of esters. The enzyme has a long and a short form. The long form is expressed in steroidogenic tissues such as testis, where it converts cholesteryl esters to free cholesterol for steroid hormone production. The short form is expressed in adipose tissue, among others, where it hydrolyzes stored triglycerides to free fatty acids.

•  HSL is activated when the body needs to mobilize energy stores, and so responds positively to catecholamines, ACTH. It is inhibited by insulin.

•  Mechanism of Activation; the first, phosphorylated perilipin A causes it to move to the surface of the lipid droplet, where it may begin hydrolyzing the lipid droplet.

•  Also, it may be activated by a PKA. This pathway is significantly less effective than the first, which is necessary for lipid mobilization in response to cyclic AMP, which itself is provided by the activation of Gs protein-coupled receptors that promote cAMP production. Examples include β-adrenergic stimulation of the glucagon receptor and ACTH stimulation of the ACTH receptor in the adrenal cortex.

Types of Blood Lipoproteins • Chylomicron (formed in cells of the small intestine) transfers dietary lipids from the intestine into the liver. • VLDL (formed in the liver) transfers lipids from the liver by blood to extrahepatic tissues. • IDL (formed in circulation) is an intermediate LP of VLDL breakdown • LDL (formed in circulation) transfers lipids from blood into tissues. • HDL (formed in the liver and small intestine) transfers lipids from extrahepatic locations to the liver. •  Hepatic lipase is expressed in the liver and adrenal glands. One of the principal functions of hepatic lipase is to convert into IDL to LDL.

•  After the binding of a ligand to plasma membrane-spanning receptors, a signal is sent through the membrane, leading to membrane coating, and formation of a membrane invagination. The receptor and its ligand are then opsonized in clathrin-coated vesicles. Once opsonized, the clathrin-coated vesicle uncoats (a pre-requisite for the vesicle to fuse with other membranes) and individual vesicles fuse to form the early endosome. Since the receptor is internalized with the ligand, the system is saturable and uptake will decline until receptors are recycled to the surface.

•  Protein C, also known as autoprothrombin IIA and blood coagulation factor XIV, is a zymogenic protein, the activated form of which plays an important role in regulating blood clotting, inflammation, cell death, and maintaining the permeability of blood vessel walls in humans and other animals. Activated protein C (APC) performs these operations primarily by proteolytically inactivating proteins Factor Va and Factor VIIIa. APC is classified as a serine protease as it contains a residue of serine in its active site. In humans, protein C is encoded by the PROC gene, which is found on chromosome 2.

Structure of rat intestinal fatty acid–binding protein. Biles salts not only aid lipid digestion but are also essential for absorption. A cytoplasmic protein in the intestinal cells, increases solubility of FA and protects cells from detergent effect of free FA’s.

Structure of human serum albumin in complex with 7 FA’s. The fasting conc of NEFA to albumin is 0.2 to 0.6mM Cytosolic FABP are found in conc of 0.1 to 1.0mM

JOURNAL OF BIOLOGICAL CHEMISTRY OCTOBER 22, 2010•VOLUME 285•NUMBER 43

FIGURE 1. Crystal structure of human FABP containing an oleic acid ligand. The protein structure is similar for all the FABP’s and shows the β-barrel domain and the N-terminal helix-turn helix motif.

When triacylglycerides are metabolized, glycerol is released, which is then metabolised to DHAP. This pathway in liver during gluconeogenesis produces NADH. Mobilization of triacylglcerides in adipose cells, is initiated by homone sensitive triacylglycerol lipase, free FA’s are relaeased into the bloodstream to bind to albumin, while glycerol is transported to the liver & kidney. The hormones epinephrine and glucagon (possibly Insulin) activate the hormone sensitive lipase.

Mechanism of fatty acid activation catalyzed by acyl-CoA synthetase. There are three isoenzymes, which a specific for chain length.

Acyl-CoA Synthases, associated with endoplasmic reticulum membranes and the outer mitochondrial membrane, catalyze activation of fatty acids, esterifying them to coenzyme A, as shown at right. This process is ATP-dependent, and occurs in 2 steps. There are different Acyl-CoA synthases for fatty acids of different chain lengths. Exergonic hydrolysis of PPi (P~P), catalyzed by pyrophosphatase, makes the coupled reaction spontaneous. Overall, two ~P bonds of ATP are cleaved during fatty acid activation. The acyl-coenzyme A product includes one "high energy" thioester linkage.

Summary of fatty acid activation: fatty acid + ATP ⇒ acyl-adenylate + PPi ⇒ 2 Pi acyladenylate + HS-CoA ⇒ acyl-CoA + AMP Overall: fatty acid + ATP + HS-CoA ⇒ acyl-CoA + AMP + 2 Pi

Acylation of carnitine catalyzed by carnitine palmitoyltransferase I. Long chain FA’s can not cross the membrane but must first form acyl-carnitine to be transported by an antiport acyl-carnitine carrier protein. Carrier takes up acyl-carnitine and exchanges it with carnitine (Acyl-carnitine/carnitine antiporter).

Transport of fatty acids into the mitochondrion. Transfer of the fatty acid moiety across the inner mitochondrial membrane involves carnitine. CPT II transfers the acyl group from carnitine to CoA in the matrix of the mitochondria. The carnitine is antiported back to the cytosol.

Malonyl-CoA inhibits Carnitine Palmitoyltransferase I. (Malonyl-CoA is also a precursor for fatty acid synthesis). Malonyl-CoA is produced from acetyl-CoA by the enzyme Acetyl-CoA Carboxylase. AMP-Activated Kinase, a sensor of cellular energy levels, catalyzes phosphorylation of Acetyl-CoA Carboxylase under conditions of high AMP (when ATP is low). Phosphorylation inhibits Acetyl-CoA Carboxylase, thereby decreasing malonyl-CoA production. The decrease in malonyl-CoA concentration releases Carnitine Palmitoyltransferase I from inhibition. The resulting increase in fatty acid oxidation generates acetyl-CoA for entry into Krebs cycle, with associated production of ATP.

The oxidation of FA yields 38kJ/g while carbohydrates and protein yield only 17kJ/g. The β-oxidation pathway of fatty acyl-CoA, this pathway in the mitochondria generates 2 ATP through complex II of the ETS & an activated FA is oxidised by 1 acyl-CoA dh, introducing an α-β trans db, 2. enoyl-H, the db is hydrated to an OH- 3. 3-HO-acyl-CoAdh, the alc is then oxidised to a ketone, formind n NADH for ETS, producing 3 ATP 4. Thiolase the acyl-CoA is cleaved to an acyl-CoA (n-2) and an acetyl-CoA.

The NADH generated by 3-hydroxyacyl-CoA dh, will feed H+ to complex I to generate 3ATP. The first three steps of the β-oxidation pathway resembles the succinate to OAA portion of the TCA cycle.

Mechanism of action of b-ketoacyl-CoA thiolase*. The over all equation for this is: L-3-HO-acyl-CoA + NAD→ 3-ketoacyl-CoA +NADH+H+ which is catalysed by hydroxyacyl CoA dh. This product the is catalysed by b-ketoacyl-CoA thiolase to form: 3-ketoacyl-CoA + HS-CoA→acetyl-CoA + acyl-CoA

There are three acyl-CoA dh; long chain, medium chain and short chain. The other enzymes are not chain length dependent.

FA oxidation is highly exergonic. Each round of βoxidation produces acetyl-CoA, NADH & FADH2. Oxidation of palmiotyl-CoA, forms: 7 FADH2,

7 NADH, 8 acetyl-CoA. Oxidation of 8 acetyl-CoA yield: 8 GTP, 24 NADH, 8 FADH2. Totals; 31 NADH= 93 ATP, 15 FADH2=30 ATP, minus 2 ATP to charge the acyl group, give a net of 129 ATP.

Oxidation of unsaturated fatty acids.Structures of two common unsaturated fatty acids. These each have specific requirements for metabolism. Odd number db are metabolised by isomerases and even number db’s are metabolised by isomerases and reductases. The first db is usually between C9 & C10. Any addition db are at 3 carbon intervals beyond.

Conversion of propionyl-CoA to succinyl-CoA. MMCoAR requires B12.

The propionyl-CoA carboxylase reaction.

The metabolic conversion of ketone bodies to acetyl-CoA. The process of ketogenesis occurs in the mitochondria of the liver. This produces a significant metabolic fuel for cardiac and skeletal muscles predominantly. During starvation hydroxybutyrate becomes the brain major metabolic source of energy. Ketone bodies are water soluble equivalents of FA. Thiolase produces acetoacetyl-CoA in the reverse of βoxidation. Condensation of a third acetyl-CoA formed by HMG-CoA synthase yields βhydroxy methylglutaryl-CoA (HMG-CoA).

If the amounts of acetyl-CoA generated in fatty-acid β-oxidation challenge the processing capacity of the TCA cycle or if activity in the TCA cycle is low due to low amounts of intermediates such as OAA, acetyl-CoA is then used instead in biosynthesis of ketone bodies via acetoacyl-CoA and HMG-CoA. Deaminated amino acids that are ketogenic, such as leucine, also feed the TCA cycle, forming acetoacetate & ACoA and thereby produce ketones.

This is the reversal of ketogenesis. In this pathway, it is the utilization of hydroxybutrate to form acetyl-CoA and feed directly into the TCA cycle.

A comparison of fatty acid β-oxidation and fatty acid biosynthesis. The biosynthesis and oxidation enzymology are the same, but a reversal of each pathway. This is the major reason for the compartmentation and control mechanisms.

Biochemistry of Peroxisomes Annu. Rev. Biochem. 2006.75:295–332

H. Rottensteiner, F.L. Theodoulou, BBA 1763 (2006) 1527–1540 Fig. 1. Transport processes associated with β-oxidation. Dependent on chain length and organism, fatty acids are imported into peroxisomes as free acids (FFA) or CoA esters (FA-CoA). CoA esters formed in the cytosol or on the endomembrane system by the action of acyl-CoA synthetases (ACS) are transported across the peroxisomal membrane by one or more ABC proteins, at the expense of ATP hydrolysis. Alternatively, short- and medium chain free acids may enter the peroxisome by passive transport and are activated by peroxisomal ACS. This reaction requires intraperoxisomal pools of ATP and CoA which are supplied by the peroxisomal adenine nucleotide translocator (ANT) and by an unknown mechanism (?) respectively. AMP and pyrophosphate (PPi) are produced as by-products of fatty acid activation; AMP is exchanged for ATP by ANT and pyrophosphate is thought to be converted to inorganic phosphate which is exported by an NEM-insensitive phosphate transport protein (PT).

Fig. 2. Transport of reducing equivalents. The dehydrogenase reaction of them ultifunctional protein of β-oxidation requires NAD+. Since the peroxisomalmembrane is impermeable to nucleotides and nucleotide-containing cofactors, two redox shuttles involved in regeneration of peroxisomal NAD+ have been proposed for plants and yeast. Both systems require exchange of organic anions across the peroxisomal membrane, which may be executed by specific carriers or porin-like proteins. (A) The malate-oxaloacetate shuttle. Peroxisomal malate dehydrogenase (MDH) converts oxaloacetate (OAA) to malate with the concomitant oxidation of NADH. Malate is exported to the cytosol, where it is re-converted to OAA by the action of cytosolic MDH. NADH produced in this reaction is re-oxidised by mitochondrial NADH dehydrogenases (NDH). Import of OAA into the peroxisome completes the shuttle. Abbreviations: ACX, acyl- CoA oxidase; MFP, multifunctional protein; thio, thiolase. (B) The malate-2- oxoglutarate shuttle. As in the malate-oxaloacetate shuttle, malate produced from OAA by peroxisomal MDH is exported from the peroxisome. Malate is then transported into mitochondria where it is converted to OAA by MDH. OAA then undergoes conversion to aspartate by aspartate amino transferase (AAT), with the concomitant formation of 2-oxoglutarate (2-OG) from glutamate. 2-OG is transferred from mitochondria to peroxisomes where the reverse reaction occurs, catalysed by peroxisomal aspartate amino transferase, to yield OAA. This shuttle also requires the transport of aspartate and glutamate between mitochondria, peroxisomes and cytosol.

CARDIAC FATTY ACID -OXIDATION Physiol Rev • VOL 90 • JANUARY 2010 FIG. 1. Overview of A β-oxidation in the heart. FA utilized for cardiac FA β-oxidation primarily originate from either plasma FA bound to albumin or from FA contained within chylomicron or very-low-density lipoproteins (VLDL) triacylglycerol (TAG). FA are taken up by the heart either via diffusion or via CD36/FATP transporters. Once inside the cytosolic compartment of the cardiac myocyte, FA (bound to FA binding proteins) are esterified to FA acyl CoA by FA acyl CoA synthase (FACS). The FA acyl CoA can then be esterified to complex lipids such as TAG, or the acyl group transferred to carnitine via carnitine palmitoyltransferase 1 (CPT-1). The acylcarnitine is then shuttled into the mitochondria, where it is converted back to fatty acyl CoA by CPT-2. The majority of this fattyacyl CoA then enters the FA β-oxidation cycle, producing acetyl CoA, NADH, and FADH2. Under certain conditions, mitochondrial thioseterase(MTE) can cleave long-chain acyl CoA to FA anions, which may leave the mitochondrial matrix via uncoupling protein.

FIG. 5. The Randle (glucose-fatty acid) cycle. The Randle cycle describes the reciprocal relationship between FA and glucose metabolism. The increased generation of acetyl CoA derived from FA β-oxidation decreases glucose (pyruvate) oxidation via the activation of PDK and the subsequent phosphorylation and inhibition of PDH. PDK is also activated by increased mitochondrial NADH/NAD ratios in response to increased FA β-oxidation. The increased supply of FA β-oxidation derived acetyl CoA to the TCA cycle can also decrease glycolysis due to the inhibitory effects of citrate [a TCA cycle intermediate which has gained access to the cytosol via the tricarboylate carrier (TCC)] on PFK-1. Citrate can also serve as a source of cytosolic acetyl CoA. The inhibition of glucose (pyruvate) oxidation is the predominant inhibitory effect of FA β-oxidation on the pathways of glucose metabolism. Conversely, the increased generation of acetyl CoA derived from glucose (pyruvate) oxidation inhibits fatty acid -oxidation, as the terminal enzyme of FA β-oxidation, 3-keto-acyl CoA thiolase, is sensitive to inhibition by acetyl CoA. Acetyl CoA derived from glucose (pyruvate) oxidation due to the activity of CAT and subsequent formation of acetyl-carnitine is also a substrate for carnitine:acetyl-carnitine transferase (CACT). CACT exports acetyl-carnitine to the cytosol, where it can be reconverted to acetyl CoA through the activity of cytosolic CAT. Cytosolic acetyl CoA is a substrate for ACC, which can increase the generation of malonyl CoA, an endogenous inhibitor of CPT I, and therefore decreases FA β-oxidation when glucose (pyruvate) oxidation is increased.