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Download Lecture 12-14 (Parker) - Department of Chemistry ::: CALTECH
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Glycolysis is the sequence of reactions that metabolize one molecule of glucose into two molecules of pyruvate with the production of two molecules of ATP Under anaerobic conditions pyruvate can be processed either to ethanol or lactate Under aerobic conditions pyruvate can be completely oxidized to CO2 In 1897 Hans and Eduard Buchner discovered that yeast extracts could rapidly ferment sucrose into alcohol. Startling discovery in its day because it was widely held that fermentation was a process that occurs within cells. This led to efforts to understand the mechanism of this extracellular fermentation. Studies in muscle extracts also revealed that lactic acid fermentation was very similar to alcohol fermentation in yeast. The complete glycolytic pathway was finally elucidated by 1940 through the pioneering contributions of Embden, Meyerhof, Neuberg, Parnas, Warburg and G&C Cori. Glycolysis is also known as the Embden-Meyerhof pathway. Glucose is an important fuel for most organisms. In mammals glucose is used by the brain under nonstarvation conditions and is the only fuel used by red blood cells. There are many different monosaccharides but glucose predominates as the primary fuel. Glucose is generated from dietary carbohydrates: Pancreatic a-amylase cleaves the 1,4 bonds of starch the products are maltose and maltotriose. Maltase cleaves maltose into two glucose molecules and aglucosidase cleaves maltotriose into three glucose molecules. Maltase and a-glucosidase are located on the surface of the small intestine these monosaccharides are now transported into the blood stream. In eukaryotic cells glycolysis takes place in the cytoplasm in two stages: Glucose enters cells through specific transport proteins after entry it is immediately phosphorylated by hexokinase and ATP to form glucose 6-phosphate. This traps the glucose into the cell because G6P cannot pass through the cell membrane and destabilizes it. Induced fit in hexokinase Hexokinase places glucose into a cleft and then the cleft closes trapping glucose and excluding H2O Conversion of G-6P into F-6P requires two steps by phosphoglucose isomerase: 1) The glucose chain must be opened 2) Isomerization to the open chain aldose to ketose F-6P 3) Then a 4 carbon atom ring closure occurs leaving closed ring F-6P A second phosphorylation step occurs after isomerization catalyzed by phosphofructokinase: Essentially non-reversible The 6-carbon F-1,6-BP is cleaved by aldolase into G3P and DHAP G3P is on a direct pathway of glycolysis whereas DHAP is not and needs to be converted into G3P by an isomerase: At equilibrium 96% of the triose phosphate is DHAP but utilization of G3P by glycolysis forces the reaction to generate G3P Structure of triose phosphate isomerase Glu165 and His95 play key roles in the reaction Glu removes the H+ from C1 and His donates a H+ to the O bound to C2 Glu165 and His95 play key roles in the reaction Glu removes the H+ from C1 and His donates a H+ to the O bound to C2 TPI traps the enediol intermediate with the adjacent loop of the enzyme preventing loss of the intermediate which would lead to the production of a toxic methyl glyoxal molecule STAGE 1 STAGE 2 Oxidation of the aldehyde to and acid powers the formation of the high phosphoryl-transfer potential 1,3-BPG Glyceraldehyde 3-P dehydrogenase synthesizes 1,3-BPG in two steps: Catalytic mechanism of glyceraldehyde 3-phosphate dehydrogenase Cys reacts with the aldehyde group of the substrate Oxidation takes place with the transfer of H+ to NAD forming a thioester intermediate NADH is replaced by NAD+ Orthophosphate attacks the thioester forming 1,3-BPG ATP is formed by phosphoryl transfer from 1,3-BPG to ADP catalyzed by PGK Additional ATP is generated with the formation of pyruvate The phosphoryl group present in the enol form generated by the action of enolase is unstable; once transferred to ADP by pyruvate kinase it rapidly undergoes conversion to the ketone pyruvate. The driving force for this reaction is large because of this conversion. Glucose+2Pi+2ADP+2NAD == 2pyruvate+2ATP+2NADH+2H+ NAD+ is regenerated from the metabolism of pyruvate Ethanol is formed from pyruvate in yeast and other microorganisms Lactate is formed in a variery of microorganisms and also in cells of higher organisms when 02 is limiting Much more energy is extracted by conversion of pyruvate to acetyl CoA via the citric acid cycle Pyruvate to ethanol regenerates NAD+ The regeneration of NAD+ by the alcohol and lactate fermentation keeps a redox balance The regeneration of NAD+ by the alcohol and lactate fermentation keeps a redox balance The three dehydrogenases that we have considered are very different structurally but all three possess a conserved NAD+ binding domain 4 enzymatic steps 1 enzymatic step Fructose in the liver enters the glycolytic pathway via the fructose 1-phosphate pathway THIS PATHWAY BYPASSES PFK NO ATP FEEDBACK TPI The fructose 1 phosphate pathway in the liver by passes ATP feed back allowing high levels of AcetylCoA to accumulate leading to the production of fatty acids and a fatty liver this occurs if consumption of high fructose corn syrup is high Galactose is converted into glucose 6-phosphate in four steps Entry of galactose into glycolysis utilizes a previously modified galactose molecule PGM Lactose intolerance is due to a low level of lactase A deficency of galactose 1-phosphate uridyl transferase can lead to toxic levels of galactose; in the eye lens galactose is converted into galactitol causing the production of a cataract The Glycolytic pathway is tightly controlled The rate of conversion of glucose to pyruvate is tightly regulated. In glycolysis the reactions catalyzed by hexokinase, phosphofructokinase and pyruvate kinase are essentially irreversible making them targets for regulation. These enzymes become more or less active in response to the binding of allosteric effectors (milliseconds) or covalent modifications (seconds). In addition the levels of these enzymes are regulated by the transcription of their genes (minutes). Phosphofructokinase is regulated by the ratio of ATP/AMP A tetramer of 4 identical subunits At high levels of ATP it will bind to an allosteric site reducing the enzyme’s affinity for fructose 6-PO AMP reverses the inhibitory action of ATP Hexokinase is inhibited by high levels of its product glucose 6-phosphate The regulation of glycolysis in the liver is more complex than in muscle tissue The liver has more diverse biochemical functions than muscle. The liver maintains blood-glucose levels and stores glucose as glycogen when glucose levels are plentiful. Phosphofructokinase regulation with respect to ATP is similar to muscle, however low pH is not a metabolic signal. In the liver phosphofructokinase is inhibited by citrate, an early intermediate in the citric acid cycle. The regulation of glycolysis in the liver is more complex than in muscle tissue A high level of citrate indicates that biosynthetic intermediates are abundant so there is no need to degrade additional glucose for this purpose. Citrate inhibits phosphofructokinase by enhancing the inhibitory effect of ATP. Glycolysis in the liver responds to changes in blood glucose through the signaling molecule; fructose 2,6-bisphosphate. F-2,6-BP is a potent activator of phosphofructokinase. In the liver the concentration of fructose 6-phosphate rises when blood glucose is high, the abundance of fructose 6-phosphate accelerates the synthesis of F-2,6-BP. F-2,6-BP increases the affinity of phosphofructokinase and diminishes the inhibitory effect of ATP. Thus glycolysis is accelerated when glucose is abundant, a process known as feed-forward stimulation. Feed-forward stimulation in response to high blood glucose levels by the liver F-2,6-BP stimulates phosphofructokinase even at low substrate concentrations (A). ATP inhibitory effects are attenuated in the presence of F-2,6-BP (B). The liver isoforms of pyruvate kinase can be modified by phosphorylation which reduce its activity and limit the livers utilization of glucose saving it for muscle and brain to consume. GLUT’s consist of a single chain of 500 amino acid isoforms: Glucose can be synthesized from non-carbohydrate precursors The gluconeogenesis pathway converts pyruvate into glucose. The major non-carbohydrate precursors are lactate, amino acids and glycerol. Lactate formed in muscle tissue and can be readily converted into pyruvate by the enzyme lactate dehydrogenase. Amino acids are derived from proteins in the diet. Glycerol is converted into dihydroxyacetone phosphate then enters the pathway via conversion to glyceraldehyde 3-phosphate Pathway of Glycolysis Pathway of Gluconeogenesis Conversion of glycerol to dihydroxyacetone phosphate Pyruvate is converted into oxaloacetate by a mitochondrial enzyme: pyruvate carboxylase Domain structure of pyruvate carboxylase Biotin-binding domain of pyruvate carboxylase; biotin is covalently attached through an e-nitrogen atom of lysine Biotin contains an activated CO2 molecule pyruvate carboxylase Malate dehydrogenase Oxaloacetate is converted into phosphoenolpyruvate by an ER bound enzyme: phosphoenolpyruvate carboxykinase Glucose 6-phosphate is converted to glucose in the ER by glucose 6-phosphatase This process occurs primarily in the liver: The stoichiometry of gluconeogenesis: 2pyruvate+4ATP+2GTP+2NADH+6H2O= glucose+4ADP+2GDP+6Pi+2NAD+2H+ DG=-11 kcal/mole Energy charge determines whether glycolysis of gluconeogenesis will be most active The most important regulatory site is the interconversion of fructose 6-phosphate and fructose 1,6-bisphosphate When energy is needed the concentration of AMP will be relatively high. AMP stimulates phosphofructokinase and inhibits fructose 1,6-bisphosphatase, glycolysis is favored. Conversely high levels of ATP and citrate indicate there are abundant biosynthetic intermediates. ATP and citrate inhibit phosphofructokinase and activate fructose 1,6-bisphosphatase, gluconeogenesis is favored. Glycolysis and gluconeogenesis are also reciprocally regulated at the interconversion of phosphoenolpyruvate and pyruvate in the liver The glycolytic enzyme pyruvate kinase is inhibited by allosteric effectors ATP and alanine, signaling energy charge is high and building blocks are abundant. Conversely pyruvate carboxylase, which catalyzes the first step of gluconeogenesis from pyruvate is inhibited by ADP. Similarly ADP inhibits phosphoenolpyruvate carboxykinase. Gluconeogenesis is favored when a cell has ample ATP and biosynthetic precursors. The balance between glycolysis and gluconeogenesis in the liver is sensitive to blood-glucose concentrations The signaling molecule fructose 2,6-bisphosphate strongly stimulates phosphofructokinase(PFK) and inhibits fructose 1,6bisphosphatase When blood-glucose is low fructose 2,6-bisphosphate looses a phosphate to form fructose 6-phosphate which can not bind to PFK. The level of fructose 2,6-bisphosphate is critical in determining whether glycolysis or gluconeogenesis will occur. Two enzymes regulate the concentration of this molecule: one phosphorylates fructose 6-phosphate and the other dephosphorylates fructose 2,6bisphosphate. Domain structure of the bifunctional enzyme phosphofructokinase 2 The activities of PFK2 and FBP2 are reciprocally controlled by the phosphorylation of a single serine residue At low glucose levels a rise in the hormone glucagon triggers a cAMP signaling cascade leading to the phosphorylation of the bifunctional enzyme by protein kinase A. This modification activates FBPase2 and inactivates PFK2 leading to a drop in the levels of F-2,6-BP resulting in gluconeogenesis. When blood glucose levels are high, insulin is secreted by the pancreas initiating a signaling pathway that activates a protein phosphatase removing the phosphoryl group from the bifunctional enzyme thus PFK2 is activated and FBP2 inhibited and glycolysis begins as the levels of F-2,6-BP rise. Promoter structure of the phosphoenolpyruvate carboxykinase gene IRE insulin response element (repression) GRE glucocorticoid response element TRE thyroid hormone response element CREI and CREII cAMP response elements (activation) The Cori cycle Lactate is converted to pyruvate by lactate dehydrogenase Isozymic forms of lactate dehydrogenase in different tissues catalyze the inter-converson of pyruvate to lactate. The two primary isoforms are H (predominates in the heart) and M (predominates in skeletal muscle and the liver). These subunits associate to form 5 types of tetramers the H4 has higher affinities for substrates than the M4. H4 oxidizes lactate to pyruvate so the heart is always aerobic. M4 is optimized to convert pyruvate into lactate to allow glycolysis to proceed under anaerobic conditions.