Both pathways start with Glucose as a substrate but they differ in the product.

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1 Glycosis:may occur either with the presence or absence of -Glucose-.So with oxygen we have Aerobic glycolysis-, without the participation of oxygen Anaerobic glycolysis-(it occur in certain places) where pyruvate is reduced to lactate as NADH is oxidized to NAD. Aerobic glycolysis :-presence of oxygen -product:2 pyruvate molecule -2 NADH mole. Produce -2 ATP mole. Synthesized Anaerobic glycolysis:-absence of oxygen -product: lactate -NO NADH production -2 ATP produced Both pathways start with Glucose as a substrate but they differ in the product. Pyruvate and lactate as a products contain most of the energy that originally contained in glucose and releasing of energy occurs by the TCA cycle that will be discuss later ) so one molecule Glucose produce molecules pyruvate, 2 ATP,2 NADH. Regulation of glycolysis: Aerobic glycolysis regulated mainly by three irreversible steps that control the critical step of, ATP consumption and ATP production (step 1,3,10) that play a significant regulatory role in glycolysis pathway. Regulatory enzymes are controlled by small molecules that can either activate or inhibit their activity. And these enzymes are: 1) Hexokinase; convert Glucose to Glucose 6 phosphate. 2) Phosphofructokinase 1(PFK1) ;covert Fructose 6 phosphate to Fructose 1,6- bisphosphate ) 1 P a g e

2 3) Pyruvate kinase ; convert Phosphoenolpyruvate to Pyruvate) The first enzyme that we mentioned before ; PFK that catalyzed the phosphorylation of fructose 6 phosphate by ATP (mainly is important regulatory enzyme cause it influenced by a wide Range of inhibitor mainly ATP and activated by ADP/AMP). Hexokinase : as a regulatory enzyme that catalyze the first step in glycolysis the phosphorylation of glucose by ATP forming Glucose-6 phosphate,it s an irreversible reaction. This enzyme inhibited by high concentration of its product Glucose 6 phosphate this inhibition known as feedback inhibition- ; the phosphorylation of glucose is inhibited if there is a buildup of glucose-6 phosphate. Glucokinase: mammals have several isozymes of the enzyme Hexokinase that catalyze the phosphorylation of glucose to glucose-6 phosphate and Glucokinase as an example. Glucokinase: is a predominant enzyme responsible for phosphorylation of glucose in LIVER. It function as a glucose sensor for controlling plasma glucose homeostasis by enhancing insulin secretion. Glucokinase Vs. hexokinase Glucokinase has an important kinetics properties : 1) Has higher Km than hexokinase ; so in normal glucose concentration (5 mmol/l) affinity to glucose is much lower than hexokinase affinity ( hexokinase has higher Km, that s indicate high affinity to bind glucose) 2) Not inhibited by Glc-6-P rather it inhibited indirectly by Fructose-6 phosphate and stimulated by glucose. High Km ; enzyme require more substrate for half saturation. 2 P a g e

3 Regulation of glucokinase: activity mediated by glucokinase regulatory protein (GKRP) that exist in nucleus of hepatocytes. From here we have two cases : High glucose level Low glucose level Increase the glucose level cause the release of GK from the regulatory protein and GK enter the cytosol where it s phosphorylates glucose to glcose- 6 phosphate. As glucose level fall,fructose-6 phosphate increase, causes GK to translocate back into nucleus and bind to RP thus inhibit the enzyme s activity Glucokinase which the body uses as a sensor for controlling plasma glucose. Our last enzyme in glycolysis Pyruvate kinase- which dephosphorylates phosphoenopyruvate- by ADP---ATP, can be found in several forms all of which are inhibited by high concentration of either ATP/Acetyl-CoA (highenergy metabolic intermediate ). Iso-enzymes: L which found in Liver M:muscle &brain. Pyruvate kinase activated by Fructose 1,6-bisphosphate ( an intermediate) also it binds phosphoenolpyruvate because it s an allosteric enzyme. Fructose-6 phosphate converted to Fructose 1,6-bisphosphate by phosphofruktokinase 1. This fructose 1,6-bisphosphate an intermediate product activate pyruvate kinase and this known as feed-forward regulation. Feed-forward: incrased phosphofructokinase activity results in elevated level of fructose 1,6-bisphosphate that will activates pyruvate kinase.(building up of an early intermediate (Fructose 1,6-bisphosphate) will regulate the last enzyme (pyruvate kinase). 3 P a g e

4 Fructose 1,6-bisphosphate is an allosteric activator to pyruvate kinase. ATP& Alanine both inhibitors to pyruvate kinase. Another way to regulate pyruvate kinase is camp protein, phosphorylation by camp-dependent protein kinase lead to inactivation of pyruvate kinase in liver. Any decreasing in glucose level will increase glycogen level so increasing glycogen level will increase the I.C levels of camp that will cause phosphorylation and inactivation of pyruvate. Dephosphorylation of pyruvate kinase by phosphorylation phosphatase is favor. Pyruvate deficiency: RBCs lack mitochondria therefore--- completely dependent on glycolysis for ATP production. ATP required for: 1. Metabolic needs for RBCs. 2. Maintain pumps that will preserve it s shape. Low ATP production will cause abnormal RBCs function (alternation in RBCs membrane lead to changes in shape ultimately death and lysis by phagocytosis mainly macrophages in spleen), this is the second common cause to hemolytic anemia(decreasing in RBCs amount) after glucose-phosphodehydogenase deficiency. Effect of elevated insulin on the intracellular concentration of fructose 2,6- bisphosphate in liver. As the doc. mentioned before, PFK1 that convert fructose 6-phosphate into fructose 1,6-bisphosphate. 4 P a g e

5 PFK1: controlled by; the available concentration of ATP/fructose-6 phoshate. Regulated by ; energy level /fructose 2,6-biphosphate. Activated by; high concentration of camp. Inhibited by; elevated level of ATP. Fructose 2,6-bisphosphate: it s a potent activator of PFK1 and is able to activate the enzyme even when ATP levels are high. It s bifunctional unit: Kinase: produce fructose 2,6-bisphosphate. Phosphatase:convert fructose 2,6----fructose 6 phosphate. High insulin level----high level of fructose 2,6bi.----kinase (inactive)---- phosphatase (active). Dephosphorylation favor. Also genetics defects of glycolytic enzyme shows deficiency in pyruvate kinase. Hormonal regulation of glycoysis: Regulation of glycolysis process may be: Allosteric activation/inhibition(regulation for short period milliseconds---sec) Phosphorylation/dephosphorylation(also short period sec----min) Transcription (last for long period hours---days) 5 P a g e The influence of these profound and slower effects will be shown as - hormonal effects- on the enzyme activity that typically occur over hours to days. Transcription :changes in genes. An increase in gene transcription may be result in changes of the hormonal states associated with the one of two either regular consumption of meals rich in carbohyadrate or administration of insulin. The increase of gene transcription resulting in increase the enzyme synthesis (regulatory enzymes), that s in well-fed state

6 Tricarboxylic acid cycle Also known as krebs cycle plays several roles in metabolism. It s the final pathway where the oxidation metabolism of carbohydrates, amino acids and fatty acids. TCA is one of those pathways known as central metabolic pathway.- The cycle occurs in mitochondria in close proximity to the reactions of electron transport chain. TCA is an aerobic pathway. Participates in number of important synthetic reactions (formation of glucose from carbon skeleton of a.a) and provides building blocks for the synthesis of some a.a and heme group. It s a cycle because its start with using oxaloacetate that reacts to form citrate and so on at the final step oxaloacetate is regenerated. So oxaloacetate at the end of the cycle is reduced. TCA cycle starts with the condensation of Acetyl-CoA from pyruvate that is the final product of glycolysis so glycolysis product is the first step in TCA cycle. Slide 2: Metabolic map that show intermediary metabolism and focus on ticcarboxlic acid cycle shown as a part of the central pathways of energy metabolism. Slide3: Show the steps of TCA, starting with Acetyl-CoA condensation with oxaloacetate (4C) to form citrate (6C) then reaction goes on with oxidation and decarboxylation and produced NADH & CO2. Slides 4/5/6: Oxidation decarboxylation of pyruvate (the end product of aerobic glycolysis). 6 P a g e

7 Fist reaction that link glycolysis to TCA cycle. An irreversible reaction that s mean our body can t reconvert Acetyl-CoA into pyruvate once pyruvate becomes Acetyl-CoA there is no back. Starting with transporting of pyruvate molecule into mitochondria matrix (which is found in the inner mitochondria membrane) and there it converts to Acetyl-CoA in the presence of an enzyme known as pyruvate dehydrogenase complex-. Pyruvate dehydrogenase complex: Large complex that might be composed of 60 subunits large molecule which can be seen with E.M. Multimolecular aggregate of 3 enzymes; pyruvate dehydrogenase(decarboxylase),dihydrolipoyl transacetylase,dihydrolipoyl dehydogenase(simply E1/E2/E3), each of these enzyme catalyzes a part of the overall reaction. Contains 5 co-enzymes that act as a carrier for the intermediates of the reactions. 1- thiamine pyrophosphate (TPP). 2-lipolic acid. 3-FAD. 4-NAD 5-CoA TPP/lipolic acid /FAD: required in catalytic amount. NAD/CoA: reactant. Regulated by two enzymes: these two enzymes activate and inactivate E1(pytuvate dehydrogenase). These two enzymes are: 1-cyclic AMP-independent PDH kinase phosphorylates.(inhibit E1) 2-PDH phosphatase.(activates E1) 7 P a g e

8 *kinase is allosterically activated by ;ATP/CoA/NADH therefore presence of these high-energy signals will turn off pyruvate dehydrogenase complex- Slides 7-11: The structure of the co-enzymes. TPP: it has an important ring that it has the active site on. The TPP act as a nucleophile with the loss of its C2 hydrogen, forming the ylide form of TPP. This ylide can then attack pyruvate, which is held by the enzyme pyruvate decarboxylase,pyruvate lose CO2 and new compound named hydroxyethyl-tpp. And this is the first step, the second step is oxidation and transfer to lipoamide that deliver Acetyl group to CoA and having Acetyl-CoA. Hdroxyethyl converted to acetyllipoamide that reduced to dihydrolipoamide and that s the third step. The last step is deoxidation of dihydrolipoamide, losing H transfer to FAD then to NAD. 8 P a g e

9 Lipoamide: thioester intermediate is that formed in the pyruvate dehydrogenase (PDH) complex as a precursor to acetyl CoA. This mode of thioester formation is coupled to reduction of an intramolecular disulfide. In PDH, an enzymelinked lipoamide accepts a hydroxyethyl fragment from thiamine pyrophosphate(tpp), forming an acetyl lipoamide. The acetyl group is subsequently transferred to coenzyme A to form acetyl CoA. Simply PDH complex reactions: The diagram below shows the reactions of the PDH complex. Starting at the left-hand side, the thiazolium form of the TPP cofactor, which is a carbanion resulting from loss of H+ from the unusually acidic C2 of the thiazole ring, attacks the carbonyl carbon of pyruvate, forming the addition compound shown at the top of the figure. This addition compound can readily undergo decarboxylation (loss of carbon dioxide), with the product hydroxyethyl-tpp stabilized by resonance. The next step is the transfer of the hydroxyethyl moiety from TPP to the oxidized form of the lipoamide cofactor. The hydroxyethyl group is electron-rich, and in its reaction with lipoamide it is in effect oxidized to the carboxyl level of oxidation, while lipoamide is reduced. This reaction can be dissected into two steps, where in the first step the electron-rich carbon atom of the hydroxyethyl group attached to TPP attacks - as a strong nucleophile - one of the relatively electron-deficient sulfur atoms of the intramolecular disulfide of oxidized lipoamide. This results in the intermediate shown at lower left in the figure, which has the form of a hemithioketal. In the next step, as TPP 9 P a g e

10 departs as a leaving group, taking electrons from the bond to the hydroxyethyl group with it, the hydroxyethyl recruits the electrons from the O-H bond, assisted by a conveniently located enzyme-derived base to accept the resulting hydrogen ion. The result of these two steps is the production of the thioester acetyl lipoamide and regeneration of the TPP cofactor. All of these reactions are catalyzed by the E1, or pyruvate dehydrogenase, component of the PDH complex. The rest of the chemistry of the PDH complex is shown at the bottom of the figure. The acetyl group is transferred from reduced lipoamide to coenzyme A (CoA) by the activity of the E2, or dihydrolipoyl transacetylase, component of the complex. This is an isoenergetic conversion of one thioester to another. The free dihydrolipoamide (reduced form of lipoamide) must be re-oxidized, and this is accomplished by the activity of E3, or dihydrolipoyl dehydrogenase, component of PDH complex. Note that the cofactor of E3 is a tightly-bound FAD molecule. The electrons from dihydrolipoamide are transferred, via FAD, to NAD+, forming NADH. This is noteworthy since in the usual order of reduction potentials, the reduction of FAD by NADH would 10 P a g e

11 the energetically favorable process. be Slide 12: Synthesis of citrate from acetyl-coa and oxaloacetate Condensation of acetyl CoA and oxaloacetate form citrate and it s the first step in TCA cycle. This reaction catalyzed by citrate synthase. It s an irreversible reaction. *octaloacetate is needed in catalytic amount (needed to the reaction but it s not consumed during the reaction.) Slide 13: 11 P a g e

12 Then citrate is isomerizes to isocitrate by aconitase with the removal of CO2 mole. 12 P a g e

13 Glycolysis over view: Done by: Doaa Massarwa 13 P a g e