Biosynthetic Pathways K. Dunlap Introduction In most living organisms, the pathways by which a compound is synthesized are usually different from the pathways by which it is degraded; two reasons are Flexibility: 1. Flexibility: If a normal biosynthetic pathway is blocked, the organism can often use the reverse of the degradation pathway for synthesis. Overcoming Le Chteliers principle: 2. Overcoming Le Chteliers principle: We can illustrate by this reaction: Introduction Phosphorylase catalyzes both the forward and reverse reactions. A large excess of phosphate would drive the reaction to the right; that is, drive the hydrolysis of glycogen. To provide an alternative pathway for the synthesis of glycogen, even in the presence of excess phosphate: Most synthetic pathways are different from the degradation pathways. Most also differ in location and in energy requirements. Carbohydrate Biosynthesis We discuss the biosynthesis of carbohydrates under two headings: Synthesis of glucose in animals and humans. Conversion of glucose to other carbohydrates. Conversion of CO 2 to carbohydrates in plants Photosynthesis takes place in plants, green algae, and cyanobacteria. Synthesis of Glucose Gluconeogenesis: Gluconeogenesis: The synthesis of glucose from noncarbohydrate sources. These sources are most commonly pyruvate, citric acid cycle intermediates, and glucogenic amino acids. Gluconeogenesis is not the exact reversal of glycolysis; that is, pyruvate to glucose does not occur by reversing the steps of glucose to pyruvate. There are three irreversible steps in glycolysis: ---Phosphoenolpyruvate to pyruvate + ATP. ---Fructose 6-phosphate to fructose 1,6-bisphosphate. ---Glucose to glucose 6-phosphate. These three steps are reversed in gluconeogenesis, but by different reactions and using different enzymes. gluconeogenesis a mechanism animals use to keep maintain blood glucose levels Takes place mainly in liver Takes place in the cytosol degradation of glycogen (glycogenolysis) is another mechanism Important precursors of glucose are 3 carbon lactate, pyruvate, glycerol and certain a.a. glycolysis and gluconeogenesis share 7 of 10 steps gluconeogenesis has 3 bypass steps bypass the irrevesible steps of glycolysis gluconeogenesis 1 st bypass: conversion of pyruvate to PEP requires 2 reactions: Pyruvate converted to oxaloacetate by action of pyruvate carboxylase using ATP oxaloacetate to PEP by action of PEP carboxykinase using GTP gluconeogenesis gluconeogenesis has 3 bypass steps bypass the irrevesible steps of glycolysis gluconeogenesis gluconeogenesis has 3 bypass steps bypass the irrevesible steps of glycolysis Other Carbohydrates Glucose is converted to other hexoses and to di-, oligo-, and polysaccharides. The common step in all of these syntheses is activation of glucose by uridine triphosphate (UTP) to form uridine diphosphate glucose (UDP- glucose) + P i. Other Carbohydrates glycogenesis: glycogenesis: The synthesis of glycogen from glucose. The biosynthesis of other di-, oligo-, and polysaccharides also uses this common activation step to form an appropriate UDP derivative. The Cori Cycle The Cori cycle. Lactate from glycolysis in muscle is transported to the liver, where gluconeogensis converts it back to glucose. Lactate dehydrogenase 2NADH 2NAD+ Lactate dehydrogenase 2NAD+ 2NADH Glycolysis Gluconeogenesis 14 Lipid Biosynthesis keystone concepts: Biosynthesis of fatty acids does not proceed as a simple reversal of fatty acid oxidation These reactions are under tight control because the process is energetically expensive Fatty acid synthesis and oxidation are coordinated and regulated together Synthesis of storage and membrane lipids from fatty acids is determined by the metabolic needs of the organism Cholesterol is synthesized from acetyl CoA and has several fates Cholesterol and other lipids are transported through the blood as lipoproteins Fatty Acid Biosynthesis While degradation of fatty acids takes place in mitochondria, the majority of fatty acid synthesis takes place in the cytosol. These two pathways have in common that they both involve acetyl CoA. Acetyl CoA is the end product of each spiral of -oxidation. Fatty acids are synthesized two carbon atoms at a time The source of these two carbons is the acetyl group of acetyl CoA. acyl carrier protein, ACP-SH. The key to fatty acid synthesis is a multienzyme complex called acyl carrier protein, ACP-SH. 16 comparison to -oxidation Different pathway Different enzymes Different parts of the cell -oxidation is in the mitochondria Fatty acid synthesis is in the cytosol Fatty Acid Biosynthesis Synthesis takes place in the cytosol Intermediates covalently linked to acyl carrier protein Activation of each acetyl CoA. acetyl CoA + CO2 Malonyl CoA Four-step repeating cycle, extension by 2-carbons / cycle Condensation Reduction Dehydration reduction Fatty Acid Biosynthesis The biosynthesis of fatty acids. ACP has a side chain that carries the growing fatty acid ACP rotates counterclockwise, and its side chain sweeps over the multienzyme system (empty spheres). 19 Activation: Irreversible formation of malonyl-CoA by acetyl-CoA carboxylase Reaction catalyzed by acetyl-CoA carboxylase Malonyl CoA Malonyl CoA is synthesized by the action of acetylCoA carboxylase. Biotin is a required cofactor. This is an irreversible reaction. Acetyl CoA carboxylation is a rate-limiting step of FA biosynthesis. AcetylCoA carboxylase is under allosteric regulation. Palmitate is a negative effector. 22 fatty acid synthase complex Multienzyme Complex with 7 different active sites 4 repeated steps include: Condensation, Reduction, Dehydration, and Reduction (NADPH electron carrier) Saturated acyl group produced is the substrate for additional rounds of the pathway Fatty Acid Synthase (FAS) FAS is a polypeptide chain with multiple domains, each with distinct enzyme activities required for fatty acid biosynthesis. ACP: Recall that CoA is used as an activator for -oxidation. For fatty acid biosynthesis, the activator is a protein called the acyl carrier protein (ACP). It is part of the FAS complex. The acyl groups get anchored to the CoA group of ACP by a thioester linkage Condensing enzyme/-ketoacyl synthase (KS). Also part of FAS, has a cysteine SH that participates in thioester linkage with the carboxylate group of the fatty acid. During FA biosynthesis, the growing FA chain alternates between K-SH and ACP-SH 24 saturated acyl group is the substrate for additional rounds of the pathway Reducing agent is NADPH Stepwise reaction 1. The acetyl group gets transferred from CoA to ACP by malonyl/acetyl-CoA-ACP transferase. 2. The acetyl (acyl) group next gets transferred to the -ketoacyl-ACP synthase (KS) of FAS complex. 3. Next, the malonyl group gets transferred from CoA to ACP by malonyl/acetyl CoA ACP transferase. This results in both arms of FAS occupied 4. The COO group of malonyl ACP is removed as CO2, the acetyl group gets transferred to the alpha carbon of malonyl ACP. This results in acetoacetyl-ACP 26 Overview of FAS Repeat cycles for elongation The result of the first cycle of fatty acid biosynthesis is a four carbon chain associated to the ACP arm. This chain gets transferred to the KS. A new malonyl CoA is introduced on the ACP arm. The reactions proceed as before. For each cycle the acyl group transferred to the malonyl CoA is 2-carbons longer the previous cycle. At the end of 7 cycles a 16 carbon chain is attached to the ACP arm (palmitoyl ACP). The C16 unit is hydrolyzed from ACP yielding free palmitate Net reaction: Acetyl CoA + 7 malonyl CoA + 14 NADPH + 14 H+ Palmitate + 7 CO2 + 8 CoA + 14 NADP+ + 6H2O Fatty Acid Biosynthesis Higher fatty acids, for example C 18 (stearic acid), are obtained by addition of one or more additional C 2 fragments by a different enzyme system. Unsaturated fatty acids are synthesized from saturated fatty acids by enzyme-catalyzed oxidation at the appropriate point on the hydrocarbon chain. 29 long chain saturated FAs are made from palmitate In the sER and mitochondria CoA is the acyl carrier Similar mechanism to FAS 30 desaturation of FAs requires a mixed-function oxidase Mammalian liver cells desaturate fatty acids on sER Mammals can only make 9 or higher fatty acids Plants can make 6 and 3 fatty acids in their sER and chloroplasts Essential Fatty Acids Mammals lack the enzymes to introduce double bonds at carbon atoms beyond C9. Hence, all fatty acids containing a double bond at positions beyond C9 have to be supplied in the diet. These are called Essential fatty acids (EFA). Linoleate (18:2 9,12) and Linolenate (18:3 9,12,15) are the two essential fatty acids in mammals. Other unsaturated fatty acids such as arachidonic acid (20:4 5,8,11,14) are derived from these two EFA. Eicosanoids are derivatives of arachidonic acid and have hormonal and signaling properties. Classified into prostaglandins, thromaoxanes and leukotrienes Membrane Lipids The two building blocks for the synthesis of membrane lipids are: Activated fatty acids in the form of their acyl CoA derivatives. Glycerol 1-phosphate, which is obtained by reduction of dihydroxyacetone phosphate (from glycolysis): Membrane Lipids Glycerol 1-phosphate combines with two acyl CoA molecules, which may be the same or different: To complete the synthesis of a phospholipid, an activated serine, choline, or ethanolamine is added to the phosphatidate by formation of a phosphoric ester. Sphingolipids and glycolipids are assembled in similar fashion from the appropriate building blocks. Cholesterol All carbon atoms of cholesterol and of all steroids synthesized from it are derived from the two-carbon acetyl group of acetyl CoA. Synthesis starts with reaction of three molecules of acetyl CoA to form the six-carbon compound 3-hydroxy-3-methylglutaryl CoA (HMG-CoA). The enzyme HMG-CoA reductase then catalyzes the reduction of the thioester group to a primary alcohol. 35 3 acetates condense mevalonate to isoprene conversion 6 isoprenes polymerize cyclization cholesterol biosynthesis 36 fates of cholesterol Synthesis in the liver Exported as: bile acids, cholesteryl esters Needed for membrane synthesis, hormone precursors, Vitamin D Insoluble in water Cholesteryl esters (CEs) are transported in lipoprotein particles or stored in the liver. 37 lipoproteins Chylomicron LDL 38 the real picture Size 1000nm 70nm 20nm 10nm 39 Major Classes of Lipoproteins Chylomicrons movement of dietary TG from intestine to other tissues where Apo CII activates LPL and uptake VLDL Excess FA and glucose is converted into hepatic TG and transported as VLDL to muscle and adipose LDL VLDL remnant after unloading TG (rich in cholesterol and CEs) delivered to extrahepatic tissues HDL protein rich and low in cholesterol and CEs. Has LCAT activity to remove cholesterol from chylomicrons and VLDL and return them to the liver 40 Amino Acids Most nonessential amino acids are synthesized from intermediates of either glycolysis or the citric acid cycle. Glutamate, for example, is synthesized by amination and reduction of -ketoglutarate, a citric acid cycle intermediate: Amino Acids Glutamate in turn serves as an intermediate in the synthesis of several amino acids by the transfer of its amino group by transamination. Amino Acids A summary of anabolism showing the role of the central metabolic pathway. citric acid cycle intermediates and some amino acids are gluconeogenic