Introduction to Metabolism

Metabolism The sum of the chemical changes that convert nutrients into energy and the chemically complex products of cells Hundreds of enzyme reactions organized into discrete pathways Substrates are transformed to products via many specific intermediates Metabolic maps portray the reactions

A Common Set of Pathways Organisms show a marked similarity in their major metabolic pathways Evidence that all life descended from a common ancestral form There is also significant diversity Autotrophs use CO 2 ; Heterotrophs use organic carbon; Phototrophs use light; Chemotrophs use Glc, inorganics use S and obtain chem energy through food generated by phototrophs.

The Sun is Energy for Life Phototrophs use light to drive synthesis of organic molecules Heterotrophs use these as building blocks CO 2, O 2, and H 2 O are recycled

Metabolism Metabolism consists of catabolism and anabolism Catabolism: degradative pathways Usually energy-yielding! “destructive metabolism” FUELS -> -> CO 2 + H 2 O + useful energy Anabolism: biosynthetic pathways energy-requiring! “constructive metabolism” Useful energy + small molecules --> complex molecules

Organization in Pathways Pathways consist of sequential steps The enzymes may be: Separate Form a multienzyme complex A membrane-bound system New research indicates that multienzyme complexes are more common than once thought

Catabolism and Anabolism Catabolic pathways converge to a few end products Anabolic pathways diverge to synthesize many biomolecules Some pathways serve both in catabolism and anabolism and are called amphibolic pathways

Digestion of food polymers:  enzyme-catalyzed hydrolysis Glycolysis:  glucose catabolism  generate ATP without consuming oxygen (anaerobic) Citric Acid Cycle:  metabolism of acetyl-CoA derived from pyruvate, fatty acids, and amino acids  acetyl oxidized to CO 2  operates under aerobic conditions  reduction of coenzymes NAD + and FAD; energy used to produce ATP Oxidative phosphorylation:  reduction of molecular oxygen by NADH and FADH 2  energy of reduced compounds used to pump protons across a cell membrane  potential energy of electrochemical gradient drives phosphorylation of ADP to ATP

Comparing Pathways Anabolic & catabolic pathways involving the same product are not the same Some steps may be common to both Others must be different - to ensure that each pathway is spontaneous This also allows regulation mechanisms to turn one pathway and the other off

METABOLIC REGULATION Regulated by controlling: 1.Amounts of enzymes 2.Catalytic activities 3.Accessibility of substrates

The ATP Cycle ATP is the energy currency of cells In phototrophs, light energy is transformed into the light energy of ATP In heterotrophs, catabolism produces ATP, which drives activities of cells ATP cycle carries energy from photosynthesis or catabolism to the energy-requiring processes of cells

Redox in Metabolism NAD + collects electrons released in catabolism Catabolism is oxidative - substrates lose electrons, usually H - ions Anabolism is reductive - NADPH provides the electrons for anabolic processes, and the substrates gain electrons

WHY ATP? Free energy is released when ATP is hydrolyzed. This energy drives reactions that need it (eg. muscle contraction) Recall coupled reactions ATP has a higher phosphoryl transfer potential

RECURRING MOTIFS IN METAB Certain compounds keep on recurring or appearing in metabolic reactions and their functions are the same in the processes Metab looks complicated but reactions are actually limited and repeating.

ACTIVATED CARRIERS These species help carry out the metabolic reactions, even nonfavorable ones, at times Example: ATP (activated carrier of phosphoryl groups)

Activated carriers of electrons for fuel oxidation: e - acceptors! Aerobic systems: O 2 is the final e - acceptor, but this does not occur directly Fuels first transfer e - to carriers: pyridine molecules or flavins. NAD + : nicotinamide adenine dinucleotide

Activated carriers of electrons for fuel oxidation: e - acceptors! FAD: Flavin adenine dinucleotide

Activated carrier of electrons for reductive biosynthesis: e - donors! NADPH: common electron donor R is phosphate group

Activated carrier of two-carbon fragments COENZYME A: carrier of acyl groups

Activated carrier of two-carbon fragments

VITAMINS Many vitamins are "coenzymes" - molecules that bring unusual chemistry to the enzyme active site Vitamins and coenzymes are classified as "water-soluble" and "fat-soluble" The water-soluble coenzymes exhibit the most interesting chemistry

Key Reactions in Metabolism

1. REDOX reactions Electron carriers are needed!

2. LIGATION reactions Bond formation facilitated by ATP cleavage

3. ISOMERIZATION reactions

4.GROUP TRANSFER

5.HYDROLYTIC reactions Bond cleavage by addition of H 2 O

6.ADDITION of functional groups to double bonds or REMOVAL of groups to form double bonds Uses lyases

GLYCOLYSIS

Glycolysis 1897: Hans and Eduard Buchner (Sucrose cell-free experiments; fermentation can take place outside of living cells) METABOLISM became simple chemistry Glycolysis: “Embden-Meyerhof pathway”

The all-important Glucose The only fuel the brain uses in non- starvation conditions The only fuel red blood cells can use WHY? Evolutionary: probably available for primitive systems

The products and their fates

AKA Embden-Meyerhof-Parnas Pathway Involves the oxidation of glucose Products: 2 Pyruvate 2 ATP 2 NADH Cytosolic Glycolysis

Anaerobic The entire process does not require O 2

Glycolysis: General Functions Provide energy in the form of ATP Generate intermediates for other pathways: Hexose monophosphate pathway Glycogen synthesis Pyruvate dehydrogenase Fatty acid synthesis Krebs’ Cycle Glycerol-phosphate (TG synthesis)

Specific functions of glycolysis Red blood cells (RBCs) Rely exclusively for energy Skeletal muscle Source of energy during exercise, particularly high intensity exercise Adipose tissue Source of glycerol-P for TG synthesis Source of acetyl-CoA for FA synthesis Liver Source of acetyl-CoA for FA synthesis Source of glycerol-P for TG synthesis

Regulation of Cellular Glucose Uptake Brain & RBC: The GLUT-1 transporter has high affinity for glucose and is always saturated. Ensures that brain and RBC always have glucose. Liver: The GLUT-2 glucose transporter has low affinity and high capacity. Uses glucose when fed at rate proportional to glucose concentration Muscle & Adipose: The GLUT-4 transporter is sensitive to insulin

Glucose Utilization Phosphorylation of glucose Commits glucose for use by that cell Energy consuming Hexokinase: muscle and other tissues Glucokinase: liver

Properties of Glucokinase and Hexokinase

Regulation of Cellular Glucose Utilization in the Liver Feeding Blood glucose concentration high GLUT-2 taking up glucose Glucokinase induced by insulin High cell glucose allows GK to phosphorylate glucose for use by liver Post-absorptive state Blood & cell glucose low GLUT-2 not taking up glucose Glucokinase not phophorylating glucose Liver not utilizing glucose during post-absorptive state

Regulation of Cellular Glucose Utilization in the Liver Starvation Blood & cell glucose concentration low GLUT-2 not taking up glucose GK synthesis repressed Glucose not used by liver during starvation

Regulation of Cellular Glucose Utilization in the Muscle Feeding and at rest High blood glucose, high insulin GLUT-4 taking up glucose HK phosphorylating glucose If glycogen stores are filled, high G6P inhibits HK, decreasing glucose utilization Starving and at rest Low blood glucose, low insulin GLUT-4 activity low HK constitutive If glycogen stores are filled, high G6P inhibits HK, decreasing glucose utilization

Regulation of Cellular Glucose Utilization in the Muscle Exercising Muscle (fed or starved) Low G6P (being used in glycolysis) No inhibition of HK High glycolysis from glycogen or blood glucose

Regulation of Glycolysis Regulation of 3 irreversible steps PFK-1 is rate limiting enzyme and primary site of regulation.

Regulation of Glycolysis Most important regulation hub!

Regulation of PFK-1 in Muscle Relatively constitutive Allosterically stimulated by AMP High glycolysis during exercise Allosterically inhibited by ATP High energy, resting or low exercise Citrate Build up from Krebs’ cycle May be from high FA beta-oxidation -> hi acetyl-CoA Energy needs low and met by fat oxidation

Regulation of PFK-1 in Liver Inducible enzyme Induced in feeding by insulin Repressed in starvation by glucagon Allosteric regulation Like muscle w/ AMP, ATP, Citrate Activated by Fructose-2,6-bisphosphate

Fermentation Anaerobic respiration! Produces ATP without oxygen. No ETC is present since there is no oxygen NAD+ gets recycled by use of an organic hydrogen acceptor like lactate or ethanol. Common in prokaryotes and very useful to humans.

Fermentation Two type lactic acid and alcohol fermentation. A build up of lactate in your muscles from over exerting yourself and not taking in enough oxygen causes soreness. Alcohol fermentation has a by product of CO2 and ethanol which is used to make alcoholic beverages. Yeast and fungus go through alcohol fermentation. The release of CO2 by yeast is what causes bread to rise.

Alcohol Fermentation pyruvate is converted to ethanol in two steps. Alcohol fermentation by yeast is used in brewing and winemaking.

Lactic Acid Fermentation pyruvate is reduced directly by NADH to form lactate Lactic acid fermentation by some fungi and bacteria is used to make cheese and yogurt The waste product, lactate, may cause muscle fatigue, but ultimately it is converted back to pyruvate in the liver.

The Tricarboxylic Acid (TCA) Cycle Also known as the Krebs Cycle and Citric Acid Cycle The citric acid cycle is the final common pathway for the oxidationof fuel molecules: amino acids, fatty acids, & carbohydrates. Most fuel molecules enter the cycle as acetyl coenzyme A This cycle is the central metabolic hub of the cell

The Tricarboxylic Acid (TCA) Cycle The citric acid cycle oxidizes two-carbon units Entry to the cycle and metabolism through it are controlled It is the gateway to aerobic metabolism for any molecule that can be transformed into an acetyl group or dicarboxylic acid, It is also an important source of precursors for building blocks

Overview of the TCA Cycle 1.The function of the cycle is the harvesting of high- energy electrons from carbon fuels 2.The cycle itself neither generates ATP nor includes O 2 as a reactant 3.Instead, it removes electrons from acetyl CoA & uses them to form NADH & FADH 2 (high-energy electron carriers) 4.In oxidative phosphorylation, electrons from reoxidation of NADH & FADH 2 flow through a series of membrane proteins (electron transport chain) to generate a proton gradient

Overview of the TCA Cycle 5.These protons then flow back through ATP synthase to generate ATP from ADP & inorganic phosphate 6.O 2 is the final electron acceptor at the end of the electron transport chain 7.The cytric acid cycle + oxidative phosphorylation provide > 95% of energy used in human aerobic cells

Fuel for the Citric Acid Cycle Thioester bond to acetate  -mercapto-ethylamine Pantothenate

70 Mitochondrion Double membrane, & cristae: invaginations of inner membrane

Oxidative decarboxilation of pyruvate, & citric acid cycle take place in the matrix, along with fatty acid oxidation Site of oxidative phosphorylation Permeable Mitochondrion

TCA Cycle: Overview Input: 2-carbon units in the form of Acetyl- CoA Output: 2 CO 2, 1 GTP, & 8 high-energy Electrons in the form of reducing elements

Cellular Respiration 8 high-energy electrons from carbon fuels Electrons reduce O 2 to generate a proton gradient ATP synthesized from proton gradient

Acetyl-CoA: Link between glycolysis and TCA Acetyl CoA is the fuel for the citric acid cycle

Pyruvate Dehydrogenase: AKA PDH The enzyme that links glycolysis with other pathways Pyruvate + CoA + NAD -> AcetylCoA + CO2 + NADH

The PDH Complex Multi-enzyme complex Three enzymes 5 co-enzymes Allows for efficient direct transfer of product from one enzyme to the next

The PDH Reaction E1: pyruvate dehydrogenase Oxidative decarboxylation of pyruvate E2: dihydrolipoyl transacetylase Transfers acetyl group from TPP to lipoic acid E3: dihydrolipoyl dehydrogenase Transfers acetly group to CoA, transfers electrons from reduced lipoic acid to produce NADH

Regulation of PDH Muscle Resting (don’t need) Hi energy state Hi NADH & AcCoA Inactivates PDH Hi ATP & NADH & AcCoA Inhibits PDH Exercising (need) Low NADH, ATP, AcCoA

Regulation of PDH Liver Fed (need to make FA) Hi energy Insulin activates PDH Starved (don’t need) Hi energy No insulin PDH inactive

Coenzymes Vitamin B 1

FAD

FADH 2

NAD

Step 1: Citrate formation Enzyme: Citrate synthase Condensation reactionHydrolysis reaction

Dehydration Hydration Step 2: Isomerization of citrate to isocitrate Enzyme: Aconitase

1st NADH produced!1st CO 2 removed Step 3: Isocitrate to α-ketoglutarate Enzyme: Isocitrate dehydrogenase

2nd NADH produced! 2nd CO 2 removed! Step 4: Succinyl-CoA formation Enzyme: α-ketoglutarate dehydrogenase

GTP produced Equivalent to ATP! GTP + ADP  GDP + ATP Step 5: Succinate formation Enzyme: Succinyl CoA synthetase

FADH 2 produced! Step 6: Succinate to Fumarate Enzyme: Succinate dehydrogenase

Step 7: Fumarate to Malate Enzyme: Fumarase

3rd NADH produced Step 8: Malate to Oxaloacetate Enzyme: Malate dehydrogenase

The TCA Cycle

Summary of the Reactions in TCA

Regulated primarily by ATP & NADH concentrations control points:  Pyruvate dehydrogenase  isocitrate dehydrogenase   - ketoglutarate dehydrogenase Control of the TCA Cycle

Biosynthetic roles of the TCA cycle

OXIDATIVE PHOSPHORYLATION

What’s the point? The point is to make ATP ! ATP

ATP accounting so far… Glycolysis  2 ATP Kreb’s cycle  2 ATP Life takes a lot of energy to run, need to extract more energy than 4 ATP! What’s the point? A working muscle recycles over 10 million ATPs per second

There is a better way! Electron Transport Chain series of molecules built into inner mitochondrial membrane along cristae transport proteins & enzymes transport of electrons down ETC linked to pumping of H + to create H + gradient yields ~30-32 ATP from 1 glucose! only in presence of O 2 (aerobic respiration) O2O2 That sounds more like it!

Mitochondria Double membrane outer membrane inner membrane highly folded cristae enzymes & transport proteins intermembrane space fluid-filled space between membranes Oooooh! Form fits function!

Electron Transport Chain Intermembrane space Mitochondrial matrix Q C NADH dehydrogenase cytochrome bc complex cytochrome c oxidase complex Inner mitochondrial membrane

G3P Glycolysis Krebs cycle 8 NADH 2 FADH 2 Remember the Electron Carriers? 4 NADH Time to break open the bank! glucose

Electron Transport Chain Intermembrane space Mitochondrial matrix Q C NADH dehydrogenase cytochrome bc complex cytochrome c oxidase complex Inner mitochondrial membrane

But what “pulls” the electrons down the ETC? electrons flow downhill to O 2 oxidative phosphorylation! O2O2

Electrons flow downhill Electrons move in steps from carrier to carrier downhill to O 2 each carrier more electronegative controlled oxidation controlled release of energy make ATP instead of fire!

H+H+ ADP + P i H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ We did it! ATP Set up a H + gradient Allow the protons to flow through ATP synthase Synthesizes ATP ADP + P i  ATP Are we there yet? “proton-motive” force

The diffusion of ions across a membrane build up of proton gradient just so H+ could flow through ATP synthase enzyme to build ATP Chemiosmosis Chemiosmosis links the Electron Transport Chain to ATP synthesis So that’s the point!

Peter Mitchell Proposed chemiosmotic hypothesis revolutionary idea at the time proton motive force True story.

H+H+ H+H+ O2O2 + Q C 32 ATP 2 Pyruvate from cytoplasm Electron transport system ATP synthase H2OH2O CO 2 Krebs cycle Intermembrane space Inner mitochondrial membrane 1. Electrons are harvested and carried to the transport system. 2. Electrons provide energy to pump protons across the membrane. 3. Oxygen joins with protons to form water. 2H + NADH Acetyl-CoA FADH 2 ATP 4. Protons diffuse back in down their concentration gradient, driving the synthesis of ATP. Mitochondrial matrix 2 1 H+H+ H+H+ O2O2 H+H+ e-e- e-e- e-e- e-e-

Cellular respiration 2 ATP~2 ATP2 ATP~34 ATP +++ ~40 ATP

Pathway Substrate-Level Phosphorylation Oxidative Phosphorylation Total ATP Glycolysis2 ATP 2 NADH = ATP CoA 2 NADH = 6 ATP6 Krebs Cycle2 ATP 6 NADH = 18 ATP 2 FADH 2 = 4 ATP 24 TOTAL4 ATP32 ATP Cellular respiration ATP

Summary of cellular respiration Oxidative phosphorylation is the process of making ATP from the reducing elements NADH and FADH 2, with the help of O 2 and the electron transport chain The electron transport chain is the structural complex that enables oxidative phosphorylation to take place

Summary of cellular respiration Where did the glucose come from? Where did the O 2 come from? Where did the CO 2 come from? Where did the CO 2 go? Where did the H 2 O come from? Where did the ATP come from? What else is produced that is not listed in this equation? Why do we breathe? C 6 H 12 O 6 6O 2 6CO 2 6H 2 O~40 ATP  +++

 ETC backs up  nothing to pull electrons down chain  NADH & FADH 2 can’t unload H  ATP production ceases  cells run out of energy  and you die! Taking it beyond… What is the final electron acceptor in Electron Transport Chain? O2O2  So what happens if O 2 unavailable? WHOA!