mahatma Phule a S C College Panvel DEPTARTMENT

mahatma Phule a. S. C. College, Panvel DEPTARTMENT OF ZOOLOGY METABOLISM BY DR. LEENA N. MESHRAM

Intermediary Metabolism All the reactions concerned with breaking down compounds and generating and storing energy for the needs of the cell and organism. All the reactions concerned with the production of compounds (metabolites) used by the cell or organism.

Introduction to Metabolism Complex substances are broken down for energy, required metabolites, structural components, etc. Cells must synthesize new complex substances. Thousands of such reactions are occurring simultaneously in a single cell.

**************************** These rxns occur with a minimum of side products , energy loss or undesired interferences and at reasonable temperatures, p. H and pressure. All of these rxns must be controlled or regulated for optimum efficiency. *****************************

****************************** Definitions: Catabolism = the breakdown of complex substances. Anabolism = the synthesis of complex substances from simpler ones. ******************************

Free Energy Changes in Metabolism -- a reminder Overall G is negative (-) for catabolic processes example: higher energy A B C D E lower energy compound G 1 compound G 2 G = G 2 - G 1 is negative

for an anabolic process, the G is positive (+) example: W V U S P -- Must supply energy, usually from ATP, to drive W P to make the overall G is negative -- So generally catabolic processes generate energy for anabolic processes ******************************

General Pathways of Metabolism -- Catabolism -1 - Breakdown of macromolecules to building blocks -- generally hydrolytic protein polysaccharide lipid nucleic acids amino glucose, glycerol ribose, het acids other sugars fatty acids bases, phosphate -- no useable energy yield hereonly building blocks obtained

2 - Breakdown of monomers to common intermediates amino glucose, glycerol, acids other sugars fatty acids pyruvate NH 4+ acetyl Co. A citric acid cycle ETS/Ox Phos ATP CO 2 Oxidative processes-- produce ATP & NADH for energy

3 -Breakdown of intermediates to CO 2 and electrons is accomplished through a central oxidative pathway: the Citric Acid Cycle or TCA or the Krebs Cycle This cycle leads to the production of ATP by processes called electron transport and oxidative phosphorylation. ******************************

proteins polysaccharides lipids amino glucose, glycerol acids other sugars fatty acids NH 4+ pyruvate acetyl Co. A Intermediates citric acid cycle --Anabolism CO 2

Anabolism, Anabolism cont’d 1 - utilization of critical Common Intermediates including components of TCA cycle to make building blocks 2 - making building block requires energy = ATP 3 - synthesis of macromolecules requires energy = ATP 4 - note CO 2 not generally reused *****************************

***************************** -- Some cells have specific nutrient requirements and cannot make some compounds, e. g. , vitamins some amino acids (about 1/2) are required in the diet by man some microorganisms cannot make certain amino acids and vitamins; these must be supplied in nature or in the media. ******************************

-- Some General Principles - Processes of metabolism are highly controlled: Anabolism and catabolism are not necessarily balanced - one or the other may predominate in certain cells or at different times depending on cell needs The pathway to synthesize a complex substance is not simply the reverse of the degradative pathway.

Modes of Control 1 - Level of energy if low, anabolism is unlikely or impossible 2 - Level of substrates 3 - Level of enzyme cofactors lipoic acid, thiamine, NAD+, etc. 4 - p. H - affects ionization states, i. e. , a molecule may be reactive only if in (un)protonated state

5 - Enzymesa) quantity- repression or induction of expression of information in DNA b) activity- may have inactive or less active states, allosteric enzymes have + or - effectors, feedback controlbuild-up of product inhibits enzyme 6 - Compartmentalization - Some enzymes and substrates restricted to certain organelles so as to make the substrate and enzyme available together in right place.

7 - Hormone control Certain cells are targeted by hormones, which indirectly regulate cellular pathways. Definition: Hormones are small regulatory molecules synthesized elsewhere and delivered to target cells.

-- One type of hormone regulates metabolism by affecting gene expression, expression e. g. , steroids. -- Another type regulates metabolism through a second messenger system Hormones act at the outside surface of the cell and cause changes in the internal levels of small molecules such as cyclic AMP, which in turn indirectly modify enzyme activities.

Carbohydrate Metabolism Overview glycogen pentose GLUCOSE other sugars pyruvate lactate acetyl Co. A TCA cycle ATP Et. OH

Enzyme Classification Dehydrogenase- oxidizes substrate using cofactors as electron acceptor or donor (pyruvate dehydrogenase) Reductase- adds electrons from some reduced cofactor (enoyl ACP reductase) Kinase- phosphorylates substrate (hexokinase) Hydrolases - uses water to cleave a molecule Phosphatase- hydrolyzes phosphate esters (glucose-6 -phosphatase) Esterase (lipase)- hydrolyzes esters (those that act on lipid esters are lipases) (lipoprotein lipase) Thioesterases - hydrolyzes thioesters Thiolase- uses thiol to assist in forming thioester (β-ketothiolase) Isomerases- interconversions of isomers (example aldose to ketose) (triose phosphate isomerase) MORE IN NOTES

GLYCOLYSIS Glucose ATP hexokinase ADP Glucose 6 -phosphate phosphoglucoisomerase Fructose 6 -phosphate ATP phosphofructokinase ADP Fructose 1, 6 -bisphosphate aldolase triose phosphate isomerase Dihydroxyacetone Glyceraldehyde phosphate 3 -phosphate

Glyceraldehyde 3 -phosphate glyceraldehyde NAD+ + Pi 3 -phosphate NADH + H+ dehydrogenase 1, 3 -Bisphoglycerate ADP phosphoglycerate kinase ATP 3 -Phosphoglycerate phosphoglyceromutase 2 -Phosphoglycerate enolase H 2 O Phosphoenolpyruvate ADP pyruvate kinase ATP Pyruvate

Pyruvate Alcohol Fermentation Anaerobic Glycolysis Aerobic Glycolysis

Glycolysis What is glycolysis? Ten step metabolic pathway to convert glucose into two molecules of pyruvate and two molecules each of NADH and ATP. All carbohydrates to be catabolized must enter the glycolytic pathway. - Glycolysis is central in generating both energy and metabolic intermediaries.

-Pyruvate can be further processed: a) anaerobically to lactate in muscle and in certain micro-organisms or b) anaerobically to ethanol (fermentation) or c) aerobically to CO 2 and H 2 O via the citric acid cycle.

Glycolysis has two stages. A. An energy investment phase. Reactions, 1 -5. Glucose to two glyceraldehyde 3 -phosphate molecules. Two ATPs are invested. B. An energy payoff phase. Reactions 6 -10. two glyceraldehyde 3 -phosphate molecules to two pyruvate plus four ATP molecules. -- A net of two ATP molecules overall plus two NADH.

Phase I. Energy Investment. 1 - Glucose is phosphorylated. Glucose enters a cell through a specific glucose transport process. It is quickly phosphorylated at the expense of an ATP. The investment of an ATP here is called “priming. ” Enzymes = hexokinase or glucokinase

ATP ADP glucose 6 -phosphate ∆Go' = -16. 7 k. J/mole

Reaction: first energy investment highly exergonic, G°´= -16. 7 k. J/mole, (essentially irreversible)

Hexokinase found in all cells of every organism low specificity for monosaccharides (simple sugars) i. e. , other monosaccharides can be phosphorylated by hexokinase. relatively high affinity for glucose, KM = 0. 1 m. M inhibited by its product, glucose 6 -phosphate

Glucokinase found in liver high KM (~10 m. M) for glucose not inhibited by glucose-6 phosphate most effective when glucose level in blood is high, i. e. , right after meal.

2 - Isomerization of glucose 6 -phosphate Enzyme = phosphoglucoisomerase glucose 6 -phosphate fructose 6 -phophate aldose to ketose isomerization reversible, G = 1. 7 k. J/mole

3 - Second phosphorylation Enzyme = phosphofructokinase ATP ADP fructose 1, 6 bisphosphate -second ATP investment -highly exergonic, essentially irreversible, G°´= -14. 2 k. J/mole - highly regulated, regulated modulating carbon flux through glycolysis in response to energy and carbon requirements

4 - Cleavage to two triose phosphates Enzyme = aldolase HC=O H 2 COP HCOH O=C HCOP + CH 2 OH H glyceraldehyde dihydroxyacetone 3 -phosphate where P = phosphate cleaves a 6 C sugar to 2 3 C sugars G°´= +23. 8 k. J/mole, driven by next rxns

-- mechanism: keto at C 2 of F-1, 6 -bis P condenses with ε-amine of lys at active site of enzyme (Schiff base intermediate), aiding in carbon-carbon bond cleavage. H H HC-OP H -H 2 O HC-OP C=O + HN C=N HCOH ENZ bond to be broken ENZ bond easily broken now

5 - Isomerization of dihydroxyacetone phosphate Enzyme = triose-phosphate isomerase H 2 C-OH C=O CH 2 -O- P dihydroxyacetone phosphate glyceraldehyde 3 -phosphate

allows interconversion of two triose phosphate products of aldolase cleavage only glyceraldehyde phosphate can be used further in glycolysis. aldose-ketose isomerization similar to phosphoglucoisomerase rxn allows dihydroxyacetone phosphate to be metabolized as glyceraldehyde 3 -phosphate reversible, G ´= +7. 5 k. J/mole. This is important in gluconeogenesis

************************* End of First Phase: - Production of two glyceraldehyde 3 -phosphate molecules from one glucose molecule with the expenditure of two ATPs. - Therefore: the energy yields of the following steps are multipled by two. ************************* Second Phase:

6 - Oxidation of glyceraldehyde 3 -phosphate Enzyme= glyceraldehyde-3 -phosphate dehydrogenase O OPO O NADH O OPOH C=O O HCOH H 2 C O- P glyceraldehyde 3 -phosphate 1, 3 bisphoglycerate + -addition of phosphate, oxidation, production of NADH, formation of high energy compound

- First high energy compound generated = beginning of payoff. - product is an acylphosphate, a fused carboxylic-phosphoric acid anhydrate, which has a very high free energy of hydrolysis. - reversible rxn, G ´ = +6. 3 k. J/mole because this fused group retains some of the energy produced by the oxidation of the aldehyde to the carboxylic acid.

-- reaction produces important reducing compound NADH = nicotinamide adenine dinucleotide, reduced form. H [Core NAD+ is recycled and not used up in metabolism. ]

Mechanism of G 3 P Dehydrogenase

7 - Transfer of phosphate to make ATP Enzyme = phosphoglycerate kinase O=C-O- P O=C-OH P HC-OH + P H 2 C-O-P P Adenosine 1, 3 PG ADP 3 -phosphoglycerate ATP - first substrate level phosphorylation, yielding ATP - 2 1, 3 bis PG yield 2 ATPs, thus so far ATP yield = ATP input - high free energy yield, G°´= -18. 8 k. J/mole drives several of the previous steps.

8 - Phosphate shift setup Enzyme= phosphoglycerate mutase - shifts phosphate from position 3 to 2 - reversible, ΔG = + 4. 6 k. J/mole

- mechanism involves phosphorylated enzyme intermediate with the formation of 2, 3 bisphoglycerate

9 - Generation of second very high energy compound by a dehydration Enzyme = enolase -- little energy change in this reaction, ΔG = +1. 7 k. J/mole because the energy is locked into enolphosphate

- the energy is locked into the high energy unfavorable enol configuration by phosphoric acid ester - upon later hydrolysis of phosphate: H high energy O -C=C low energy O -C-C- This energy is recovered the next step.

10 - Final generation of ATP Enzyme = pyruvate kinase P O H ADP ATP O - OOC-C=CH - OOC-C-CH 3 phosphoenolpyruvate - second substrate level phosphorylation yielding ATP - highly exergonic reaction, irreversible, ΔG = -31. 4 k. J/mole.

- rxn is so exergonic because the enol in PEP is transformed to a keto in pyruvate. - drives several previous reactions. - pyruvate is the primary product of glycolysis - pyruvate kinase is a highly regulated enzyme.

Bookkeeping: Bookkeeping - 2 ATPs from each glyceraldehyde 3 -phosphate = total of 4 per original glucose in second phase. - 2 molecules of NADH also produced. - 2 ATPs were invested in the first phase of glycolysis. Glycolysis: Invest 2 ATP 4 ATP net 2 ATP and 2 NADH

Summary of Energy Relationships for Glycolysis Input = 2 ATP 1. glucose + ATP glucose-6 -P 2. fructose-6 -P + ATP fructose 1, 6 bisphosphate Output = 4 ATP + 2 NADH 1. 2 glyceraldehyde 3 -P + 2 Pi + 2 NAD+ 2 (1, 3 bisphoglycerate) + 2 NADH 2. 2 (1, 3 bisphoglycerate) + 2 ADP 2 (3 -P-glycerate) + 2 ATP 3. 2 PEP + 2 ADP 2 pyruvate + 2 ATP Net = 2 ATP and 2 NADH

Energy Yield From Glycolysis glucose 6 CO 2 = -2840 k. J/mole 2 ATPs produced = 2 x 30. 5 = 61 k. J/mole glucose Energy yield = 61/2840 = 2% recovered as ATP - subsequent oxidation of pyruvate and NADH can recover more of the free energy from glucose.

Fate of Product of Glycolysis- Pyruvate is at a central branch point in metabolism. Recall: Aerobic pathway - through citric acid cycle and respiration; this pathway yields far more energy and will be discussed later. NADH + O 2 NAD+ + energy Pyruvate + O 2 3 CO 2 + energy

Two anerobic pathways: - to lactate via lactate dehydrogenase - to ethanol via ethanol dehydrogenase - Note: both use up NADH produced so only 2 ATP per glucose consumed

1. Lactate Fermentation Enzyme = Lactate Dehydrogenase COOCOOC=O + NADH + H+ H-C-OH + NAD+ CH 3 pyruvate lactate - Note: uses up all the NADH (reducing equivalents) produced in glycolysis.

Helps drive glycolysis by using up NADH Reversible so pyruvate can be regenerated in alternative metabolism Lactate fermentation important in red blood cells, parts of the retina, and in skeletal muscle cells during strenuous exercise. Important in plants and in microbes growing in absence of O 2.

-- Lactate Dehydrogenase (LDH)has multiple forms. It is an isozyme. Two polypeptides M and H come together to form LDH. It is a tetramer so a mixture is formed: M 4, M 3 H, M 2 H 2, MH 3 and H 4 M M M H H M H H H

Skeletal muscle and liver contain predominantly the “M” forms; heart the “H” forms. During and after myocardial infarction (heart attack), heart cells die releasing LDH into the circulation. Diagnostic.

LACTIC ACID (CORI) CYCLE glucose-6 -P glucose-6 -P glycogen ATP NADH Blood NADH pyruvate lactate Liver Muscle

The liver uses most of this lactate to make glycogen. Only small amounts of free glucose released. Glycogen can be broken down into glucose when needed.

2. Alcoholic Fermentation COOCO 2 CH 2 OH H O C=O C + NADH CH 3 + CH 3 NAD+ pyruvate ethanol acetaldehyde pyruvate decarboxylase-irreversible alcohol dehydrogenase- reversible

- pathway is active in yeast. - second step helps drive glycolysis -second step is reversible - reverse is ethanol oxidation, eventially yields acetate, which ultimately goes into fat synthesis. - ethanol acetaldehyde acetate - humans have alcohol dehydrogenase in liver which mainly disposes of ethanol. - acetaldehyde is reactive and toxic.

Summary Glucose of Reactions 2 ATP 2 NADH 2 pyruvate 2 NADH anaerobic 2 ethanol + CO 2 2 NADH anaerobic 2 lactate 2 acetyl Co. A + 2 CO 2 aerobic 4 CO 2 + 4 H 2 O

-- REGULATION OF GLYCOLYSIS -Three irreversible kinase reactions primarily drive glycolysis forward. hexokinase or glucokinase phosphofructokinase pyruvate kinase These enzymes regulate glycolysis as well.

1. HEXOKINASE and GLUCOKINASE – Discussed HEXOKINASE Phosphorylation of glucose. Inhibited by its product, glucose 6 -phosphate, as a response to slowing of glycolysis

GLUCOKINASE liver enzyme with high KM for glucose so most effective when glucose levels are very high not inhibited by glucose 6 -phosphate sensitive to high glucose in circulation from recent meal it decreases high level of glucose in blood by taking glucose into liver

2. PHOSPHOFRUCTOKINASE rate limiting for glycolysis an allosteric multimeric regulatory enzyme. Measures adequacy of energy levels. Inhibitors: ATP and citrate high energy Activators: ADP, AMP, low energy and fructose 2, 6 bisphosphate

ATP inhibits phosphofructose activity by decreasing fructose 6 -phosphate binding AMP and ADP reverse ATP inhibition Fructose 2, 6 bisphosphate is a very important regulator, controlling the relative flux of carbon through glycolysis versus gluconeogenesis. - It also couples these pathways to hormonal regulation.

3. PYRUVATE KINASE PEP + ADP pyruvate + ATP An allosteric tetramer - inhibitor: ATP - inhibitors: acetyl Co. A and fatty acids (alternative fuels for TCA cycle) - activator: fructose 1, 6 bisphosphate (“feed-forward”)

Phosphorylation (inactive form) and dephosphorylation (active form) under hormone control. Also highly regulated at the level of gene expression (“carbohydrate loading”)