Dr. Pritchard Gluconeogenesis Proof: Coty Cantrell

Funamentals I: 1st Hour 1 August 27, 2010 Scribe: Abby Northcutt Dr. Pritchard Gluconeogenesis Proof: Coty Cantrell

I. Gluconeogenesis (S1) a. Reverse of glycolysis b. 160g = ½ cup of sugar c. Major organ of use = brain c.i. Billions of neurons must maintain electrical potential which requires lots of ATP c.i.1. Make ATP via glycolysis of sugar then the TCA cycle c.ii. Kidney also uses a lot of glucose b/c it’s constantly filtering blood & reabsorbing molecules d. Must get glucose in diet or make it d.i. Made from proteins d.ii. If you don’t have proteins or glucose body will break down muscle d.ii.1. People that are on low carb diets don’t get enough nutritional glucose so have to make it from protein d.ii.2. Muscle broken down into protein then into glucose d.iii. Humans can not convert fat to glucose – plants can though II. Historical Perspective (S2) a. Fermentation can happen w/o living cells present b. Brothers wanted to sell yeast for its health properties (has lots of vitamins), but had problems preserving it a.i. Couldn’t preserve it w/salt b/c they couldn’t get the salt out a.ii. Before freeze drying & no one had a freezer to keep it cold a.iii. Tried using sugar but yeast started fermenting the sugar via anaerobic glycolysis a.iii.1. At really high sugar concentrations bacteria can’t grow a.iii.1.a. But it didn’t kill the yeast a.iii.2. Yeast smelled of alcohol and foamed up a.iv. Anaerobic glycolysis = fermentation III. Historical Perspective (cont’d) (S3) a. Must have phosphate for fermentation b. Divided yeast into 2 fractions using a porcelain filter a.i. Zymase: Large molecules got stuck in filter a.ii. Cozymase: Small molecules went through filter a.ii.1. Ex. Acetyl-CoA, phosphate, ATP, etc. c. almost all enzymes found in muscles except alcohol dehydrogenase are also found in yeast IV. Enzymatic Differences between Glycolysis & Gluconeogenesis (S4) a. 7 of the 10 enzymes in glycolysis are the exact same as enzymes used in gluconeogenesis – it’s easier to remember the differences V. First Reaction of Gluconeogenesis (S5) a. pyruvate = end product of glycolysis & starting molecule of gluconeogenesis b. want to convert pyruvate back into glucose c. bicarbonate = buffer in blood 2 d. uses ATP VI. Biotin & Avidin (S6) a. Pyruvate Carboxylase a.i. Carboxylase = enzyme that adds CO2 to a molecule a.ii. Uses Vitamin cofactor biotin a.ii.1. Biotin: two 5 member rings a.ii.1.a. One ring has a sulfur a.ii.1.b. Alkyl chain has an amide link to an ester b. CO2 is added then flexible chain moves to another area of the enzyme to carry out the reaction VII. Pyruvate Carboxylase Mechanism (S7) a. Takes bicorbonate & makes carbonyl phosphate a.i. Carbonyl group is linked to a phosphate b. transfers CO2 to N forming molecule on the right c. Biotin is covalently bound to enzyme d. Enzyme also binds pyruvate & deprotonates it a.i. Adds CO2 to pyruvate resulting in oxaloacetate e. Process requires energy (ATP) f. Takes place in mitochondria!!! – very easy exam question a.i. Pyruvate Carboxylase reaction is the only one – all other reactions of gluconeogenesis take place in cytoplasm VIII. Pyruvate to Oxaloacetate (S8) a. Large molecules can’t just diffuse through mitochondrial membrane b. Oxaloacetate must be reduced by NADH to malate to cross the mitochondrial membrane a.i. Once it’s out it is reoxydized into oxaloacetate a.ii. Process called shuttling IX. PEP Carboxykinase Reaction (S9) a. Second reaction of gluconeogenesis b. Cleaves CO2 & attaches a phosphate to oxaloacetate c. Called a kinase b/c ATP is involved d. Single ATP can’t add phosphate to pyruvate to make PEP b/c it requires more energy than the ATP has a.i. Doing it this way is more energetically favorable (cutting off the CO2) a.ii. Jesse asks a question in his deep burley voice a.iii. 1 ATP can cleave the CO2 from oxaloacetate b/c it’s energetically favorable a.iv. follows this path so it can use only 2 ATP to make a higher energy molecule of PEP a.iv.1.a. P EP’s phosphate bond is higher energy than ATP’s a.iv.1.b. You have to use the energy of 2 ATPs to make it e. GTP is almost the same as ATP & can be interconverted X. S10 – Skipped XI. Gluconeogenesis / Glycolysis Reaction Pathway (S11) a. Read from the bottom up for Gluconeogenesis b. Same enzymes used in glycolysis & gluconeogenesis for most reactions Funamentals I: 1st Hour 3 August 27, 2010 Scribe: Abby Northcutt Dr. Pritchard Gluconeogenesis Proof: Coty Cantrell a.i. Shown w/double arrow c. 4 starred reactions are enzymes unique to gluconeogenesis a.i. have one way arrow XII. Enolase Reaction (S12) a. Read reactions backwards! b. Enolase adds water to PEP to make 2-Phosphoglycerate c. Same enzyme is in glycolysis XIII.Phosphoglycerate Mutase Reaction (S13) a. Mutase = enzyme that moves a functional group (like a phosphate) from one place to another a.i. Moves phosphate from 2 position to 3 position b. Also used in glycolysis XIV. Isomerase, Kinase, & Mutase (S14) a. Kinase: ATP must be involved in adding or cleaving phosphate a.i. Can still cleave or add w/o ATP but that’s not a kinase b. Isomerase: converts an aldose to a ketose or vice versa XV. Phosphoglycerate Kinase (S15) a. Use ATP to add phosphate to carboxylic acid group on 3-Phosphoglycerate to form 1,3-Bisphosphoglycerate a.i. Kinase: ATP must be involved XVI. Glyceraldehyde-3-Phosphate Dehydrogenase Reaction (S16) a. Carboxylic acid attached to phosphate = mixed anhydride b. NADH reduces carboxylic acid group on 1,3-Bisphosphoglycerate to a carbonyl aldehyde c. Phosphate is also split off d. This is not a kinase b/c no ATP is involved XVII. Triose Phosphate Isomerase (S17) a. G-3-P is interconverted with Dihydroxy-Acetone-Phosphate b. Favorite of biochemists b/c closest thing to a perfect enzyme a.i. Tiny amounts catalyze this reaction perfectly c. Glyceraldehyde-3-Phosphate just bumps into enzyme & gets converted very quickly d. In glycolysis fructose-1,6-bisphosphate is cleaved into these two molecules by aldolase e. Aldolase can also rejoin these two to form fructose-1,6-bisphosphate XVIII. Gluconeogenesis / Glycolysis Reaction Pathway (S18) a. We are at the horizontal bar half way up XIX. Aldolase (S19) a. Aldol Cleavage: split molecules apart; in glycolysis b. Aldol Condensation: join together; in gluconeogenesis XX. Aldol Condensation (S20) a. Aldol = beta hydroxy carbonyl compound a.i. Aldol = Molecule with carbonyl group & a hydroxyl group two C’s away a.i.1. Can be cleaved b. Normally very hard to break C-C bonds, but can be done w/specific enzyme 4 a.i. Works at body temperature, don’t have to have strong acid or 500 degree temperatures XXI. Aldolase Structure (S21) a. Widely studied – exact structure is known a.i. Scientists know exactly where the two trioses bind XXII. Gluconeogenesis / Glycolysis Reaction Pathway (S22) a. We’re at Fructose-1,6-Bisphosphate – next step is unique to gluconeogenesis a.i. Opposite of key reaction in glycolysis – phosphofructokinase (PFK) reaction b. Takes phosphate off of fructose-1,6-bisphosphate to make fructose-6-phosphate XXIII. Fructose-1,6-Bisphosphate (S23) a. Reverse reaction requires ATP to add the phosphate b. Phosphate is cleaved off to yield fructose-6-phosphate XXIV. Phosphoglucose Isomerase (S24) a. Converts fructose-6-phosphate to glucose-6-phosphate b. Ketose converted to aldose a.i. Characteristic of an isomerase XXV. Phosphorylation of Glucose (Transport) (S25) a. when cell takes up glucose, Hexokinase phosphorylates it to lock it inside the cell a.i. there are many glucose transporters controlled by insulin that can transport glucose in or out, but once it’s phosphorylated it can’t get through the membrane a.ii. it’s not phosphorylated in liver or kidney b/c these organs are responsible for gluconeogenesis a.ii.1. only these two organs can do gluconeogenesis XXVI. Glucose-6-Phosphate (S26) a. In the liver cell endoplasmic reticulum there is an enzyme called glucose-6- phosphatase a.i. This enzyme is only found in liver & kidney in membrane of ER a.ii. Takes up glucose-6-phosphate & dephosphorylates it a.ii.1. The glucose is dumped into the lumen of the ER a.ii.2. Glucose is packaged into vesicles & bud off ER, fuse to plasma membrane, & dump into bloodstream XXVII. Cori Cycle (S27) a. When you exercise vigorously muscles don’t get enough oxygen to break down all the pyruvate that is formed, so pyruvate is converted to lactic acid (lactate) a.i. Break down takes lots of oxygen a.ii. We need to be able to reconvert NAD to NADH b. Anaerobic glycolysis results in formation of lactic acid (lactate) a.i. Lactate travels through blood stream to liver which converts it back to pyruvate & then to glucose a.ii. Then it’s dumped back into the bloodstream & goes back to muscles XXVIII. Regulation of Gluconeogenesis (S28) a. Occurs in liver b. High levels of glucose-6-phosphate cause rate to increase greatly c. Reciprocally regulated: if you activate glycolysis you will simultaneously inhibit gluconeogenesis & vice-versa a.i. Can’t have glycolysis and gluconeogenesis at the same time Funamentals I: 1st Hour 5 August 27, 2010 Scribe: Abby Northcutt Dr. Pritchard Gluconeogenesis Proof: Coty Cantrell a.i.1. You’d just be wasting ATP XXIX. Fructose-2,6-Bisphosphate (S29) a. Not an intermediate metabolite – it’s a regulatory molecule a.i. Only tiny amounts made a.ii. Affects enzymes of glycolysis & gluconeogenesis b. F-2,6-BP activates phosphofructokinase which is the key enzyme of glycolysis a.i. Speeds up glycolysis & inhibits gluconeogenesis c. In glycolysis there is Fructose-1,6-Bisphosphate which IS an enzyme XXX. Fructose-2,6-Bisphosphate Diagram (S30) a. Bifunctional Enzyme makes F-2,6-BP XXXI. 6-Phosphofructo-2-Kinase / Fructose-2,6-Bisphosphate (S31) a. One domain puts extra phosphate in 2 position of F-6-P b. The other domain removes the Phosphate XXXII. 6-Phosphofructo-2-Kinase / Fructose-2,6-Bisphosphatase (S32) a. If regulatory domain is phosphorylated the kinase domain is inhibited and the phosphatase domain is activated b. If you dephosphorylate the regulatory region the kinase domain is activated and the phosphatase domain is inhibited a.i. Enzyme function is regulated by phosphorylation a.ii. Determines whether F-2,6-BP is made or broken down c. When blood glucose is low body dumps out glucagon a.i. Glucagon increases phosphorylation a.ii. Phosphorylation inhibits the kinase domain  results in down regulation of glycolysis a.iii. When you have low glucose you don’t want to break it down you want to make it – makes sense d. If you have high glucose you will want to activate glycolysis XXXIII. Regulation of Glycolysis & Gluconeogenesis (S33) a. “Go through this yourself; all the steps make sense.” …Thanks Pritchard b. + = step activator c. - = step inhibitor d. “Ask yourself what makes sense for low and high sugar concentrations.” XXXIV. Substrates for Gluconeogenesis (S34) a. Good Exam Question, “What is not a substrate of gluconeogenesis?” a.i. Can NOT use fat to make sugar!!! – made this point many times a.i.1. Fatty acids are broken down into Acetyl-CoA which we can’t use to make glucose b. Glucogenic = amino acids that can be converted into glucose (most AA’s) c. Ketogenic = amino acids that can only be converted into ketone (Lysine & Leucine) XXXV. Citric Acid Cylce Diagram (S35) a. Why can’t you convert fatty acids to glucose? a.i. Acety-CoA has 2 carbons that come in and 2 that go out so you never end up making more oxaloacetate 6 a.ii. No matter how much you put in you get the same amount of oxaloacetate b. You can use excess oxaloacetate to make sugars a.i. Excess is made by breaking down amino acids a.ii. Amino acids enter cycle as intermediate substrates a.ii.1. Alpha- ketoglutarate, succinyl-CoA, Fumarate, or even oxaloacetate XXXVI. Plants and Bacteria – Glyoxylate Cycle (S36) a. Isocitrate ligase & Malate Synthase a.i. Enzymes that allow plants to reverse acetyl-CoA formation a.ii. Humans don’t have these enzymes a.iii. Create shunt across Citric Acid Cycle by forming Glyoxylate b. Glyoxylate: consists of a carboxylic acid group and a carbonyl group c. Isocitrate Ligase: breaks Isocitrate into Glyoxylate & Succinate a.i. Succinate can continue on to become fumarate a.ii. Glyoxylate is added to Acetyl-CoA to become Malate d. Malate is oxidized to form Oxaloacetate XXXVII. Glyoxylate Cycle vs TCA Cycle (S37) a. shows similarity b/w cycles XXXVIII. Overview of Products and Enzymes of Gluconeogenesis (S38) a. Reiterating concept XXXIX. Enzymatic Differences b/w Glycolysis & Gluconeogenesis (S39) a. Remember differences b/w glycolysis & gluconeogenesis – especially the unique enzymes b. Three on left are unique to glycolysis & Four on right are unique to gluconeogenesis a.i. The other Seven are identical

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