METABOLISM Robert J Cohen Ph.D.

Cohen,
ARB-- R3-206B - 392-4050 Email: rjcohen@ufl.edu Office Hours: MTWF 3-4pm Web page:
www.med.ufl.edu/biochem/rcohen/rcohen.html

jReadings in Lehninger, 5th edition j Lehninger, pages

Overview: 485-488, 569-577 Glycolysis: Glycolysis: 527-551, 584, 588-589 Gluconeogenesis: Gluconeogenesis: 551-558, 582-594 Glycogen: 246, 594-609 TCA Cycle: 615-638 Pentose Phosphate Shunt: 558-564 ETS: 707-722, 512-521 ATP synthesis: 723-737 ===============================

Metabolite = Biological compound Metabolite Catabolism: All the reactions Catabolism: concerned with breaking down compounds and generating and storing energy for the needs of the cell and organism. Energy = ATP Anabolism: All the reactions Anabolism: concerned with the biosynthesis of complex compounds from simpler compounds. Usually use ATP.

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 NH4+ acetyl CoA citric acid cycleETS/Ox PhosATP CO2 Oxidative processes-- produce ATP & NADH for energy

3-Breakdown of intermediates to CO2 and electrons is accomplished through a central oxidative pathway:  the Citric Acid Cycle or TCA or the Krebs Cycle Cycle.  This cycle leads to the production of ATP by processes called electron transport and oxidative phosphorylation.
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--

--

proteins polysaccharides lipids amino glucose, glycerol acids other sugars fatty acids NH4+ pyruvate Common acetyl CoA Intermediates citric acid cycle CO2

, contd 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 CO2 not generally reused *****************************************

Modes of Control- Regulation Control1- Level of energy if low, anabolism energyis unlikely or impossible 2- Level of substrates 3- Level of enzyme cofactors cofactorslipoic acid, thiamine, NAD+, etc. 4- EnzymesEnzymesa) quantity repression or induction quantityof expression of information in DNA

b) activity may have inactive or less activityactive states, allosteric enzymes have activators(+) or inhibitors(-) Example: feedback control-build-up of final product inhibits enzyme -Covalent modifications 5- Compartmentalization - Some enzymes and substrates restricted to certain organelles so as to make the substrate and enzyme available together in right place.

6- Hormone control Certain cells controlare targeted by hormones, which indirectly regulate cellular pathways. Definition: Hormones are small regulatory molecules synthesized elsewhere and delivered to target cells; they then regulate specific aspects of their metabolism.

-- One type of hormone regulates metabolism by affecting gene expression, expression e.g., steroids. -- Another type regulates metabolism system. 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 pyruvate lactate acetyl CoA TCA cycle
ETS

other sugars

EtOH ATP

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 ( -ketothiolase) Isomerases- interconversions of isomers (example aldose to ketose) (triose phosphate isomerase) Mutase- shifting groups from one part of molecule to another (phosphoglucomutase) Carboxylase- adds carboxyl group (pyruvate carboxylase) Decarboxylase- removes carboxyl group (pyruvate decarboxylase) Oxidases- uses molecular oxygen as electron acceptor (cytochrome c oxidase)

Enzyme Classification-Contd
Hydroxylase- adds hydroxyl group (steroid hydroxylases) Transferase- transfers chemical group from one molecule to another or within same molecule (fatty acyl transferase) Hydrase (hydratase)- adds water to double bond (enoyl CoA hydrase) Dehydrase (dehydratase)- removes water ( -hydroxyacyl ACP dehydratase) (when making enol, enolase) Lyase- splits a molecule often with electronic rearrangment but no water involvement (citrate lyase) Ligase- creates a new chemical bond joining two molecules with electronic rearrangement and no water involvement Phosphorylase- cleaves bond with incorporation of phosphate into the product (glycogen phosphorylase) Pyrophosphorylase- cleaves bond yielding pyrophosphate as one of the products (UDPG pyrophosphorylase) Synthetase- synthesizes product using nucleotide triphosphate (ATP) as substrate (fatty acyl CoA synthetase) Synthase- synthesizes product without ATP requirement (HMGCoA synthase)

GLYCOLYSIS 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-Bisphosphoglycerate ADP phosphoglycerate kinase ATP 3-Phosphoglycerate phosphoglyceromutase 2-Phosphoglycerate enolase H2O Phosphoenolpyruvate ADP pyruvate kinase ATP

Pyruvate

Glycolysis
What is glycolysis?  Purpose- a metabolic pathway to convert one molecule of glucose into 2 molecules of pyruvate and produce 2 molecules each ofNADH and ATP.  All carbohydrates to be catabolized must enter the glycolytic pathway.  Glycolysis is central in generating both energy and metabolic intermediaries.

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

glucose 6-phosphate G ' = -16.7 kJ/mole

o

Reaction:
 first energy investment  highly exergonic, (G = -16.7 kJ/mole, (essentially irreversible)

Hexokinase  found in all cells of every organism  relatively high affinity for glucose, KM = 0.1 mM  low specificity for monosaccharides (simple sugars) i.e., other monosaccharides can be phosphorylated by hexokinase.  inhibited by its product, glucose 6-phosphate

 found in liver  Low affinity KM (~10mM) for glucose  not inhibited by glucose-6phosphate  most effective when glucose level in blood is high, i.e., right after meal.  It quickly diminishes glucose levels in blood stream to normal by taking glucose into liver.

2- Isomerization of glucose 6-phosphate Enzyme = phosphoglucoisomerase

glucose 6-phosphate fructose 6-phosphate

aldose to ketose isomerization reversible, (Grd= 1.7 kJ/mole

3- Second phosphorylation Enzyme = phosphofructokinase

ATP ADP fructose 1,6 bisphosphate

-second ATP investment -highly exergonic, essentially irreversible, (G= -14.2 kJ/mole - highly regulated modulating carbon regulated, flux through glycolysis in response to energy and carbon requirements

4- Cleavage to two triose phosphates Enzyme = aldolase

HC=O H2COP HCOH O=C HCOP + CH2OH H
glyceraldehyde dihydroxyacetone 3-phosphate phosphate
where P = phosphate

 cleaves a 6C sugar to 2 3C sugars  (G = +23.8 kJ/mole, driven by one of the following rxns

5-Isomerization of Dihydroxyacetone phosphate

Enzyme = triose-phosphate isomerase trioseH2C-OH C=O CH2-O- P

dihydroxyacetone phosphate glyceraldehyde 3-phosphate

allows interconversion of two triose phosphate products of aldolase cleavage only glyceraldehyde 3-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 kJ/mole. This is important in gluconeogenesis.

****************************************  Production of two glyceraldehyde 3-phosphate molecules from one glucose molecule with the expenditure of two ATPs.  Therefore: the product & energy yields of the following steps are multiplied by two relative to glucose. *****************************************

6- Oxidation of glyceraldehyde 3-phosphate Enzyme= glyceraldehyde-3-phosphate glyceraldehydedehydrogenase O

HOPO OH NAD NADH O OPOH C=O OHCOH H 2C O- P glyceraldehyde 3-phosphate 1,3 bisphosphoglycerate

-addition of phosphate, oxidation, production of NADH, formation of high energy phosphorylated 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 kJ/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, oxidized and H reduced form

[Note: core NAD+ is recycled and not used up in metabolism.]

Mechanism of G3P Dehydrogenase

7- Transfer phosphate to make ATP Enzyme = phosphoglycerate kinase

O=C-O- P O=C-OH P HC-OH + P HC-OH + P H2C-O-P P H2C-O-P P Adenosine Adenosine

1,3PG ADP 3-phosphoglycerate ATP - first substrate level phosphorylation, yielding ATP - 2 1,3 bisPG yield 2 ATPs - high free energy yield, (G= -18.8kJ/mole drives several of the previous steps.

8- Phosphate shift setup Enzyme= phosphoglycerate mutase

- shifts phosphate from position 3 to 2 - reversible, Grd = + 4.6 kJ/mole

9- Generation of second very high energy compound by a dehydration Enzyme = enolase

Grd = +1.7 kJ/mole Enolphosphate is a high energy bond

10- Final generation of ATP Enzyme = pyruvate kinase P O H ADP ATP O -OOC-C=CH -OOC-C-CH 3
phosphoenolpyruvate pyruvate

- second substrate level phosphorylation yielding ATP - highly exergonic reaction, irreversible, Grd = -31.4 kJ/mole.

- drives several previous reactions. - pyruvate is the primary product of glycolysis. Pyruvate is at a central branch point in metabolism and can be used in several ways. - pyruvate kinase is a highly regulated enzyme. End of pathway

- 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

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 bisphosphoglycerate) + 2 NADH 2. 2 (1,3 bisphosphoglycerate) + 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 CO2 = -2840 kJ/mole 2 ATPs produced = 2 x 30.5 = 61 kJ/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.

Pyruvate Alcohol Fermentation Lactic Acid Fermentation

Aerobic Glycolysis Fate of Pyruvate

-Pyruvate can be further processed: Pyruvate a) aerobically to CO2 and H2O via the citric acid cycle. This yields far more energy in the form of ATP. b) anaerobically to lactic acid (lactate) in muscle and in certain micro-organisms or c) anaerobically to ethanol (fermentation) or

1. Lactate Fermentation Enzyme = Lactate Dehydrogenase COOCOO+ + C=O + NADH + H ' H-C-OH + NAD CH3 CH3

pyruvate

lactate

- Note: uses up all the NADH (reducing equivalents) produced in glycolysis.

 Helps drive glycolysis by using up NADH, making NAD+  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 O2.

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

Liver

Muscle

 The liver releases some of the glucose.  The liver uses most of this lactate to make glycogen.  Glycogen can be broken down into glucose when needed.

2. Alcoholic Fermentation COOC=O CH3 CO2 H O C + NADH CH3 CH2OH CH3 + NAD+

pyruvate

acetaldehyde

ethanol

pyruvate decarboxylase-irreversible alcohol dehydrogenase- reversible Note: NADH used up

- pathway is active in yeast. - 2nd step helps drive glycolysis -2nd step is reversible - humans have alcohol dehydrogenase in liver, which is mainly used to dispose of ethanol. - reverse of 2nd step is ethanol oxidation, eventially yields acetate, which ultimately goes into fat synthesis. - ethanol acetaldehyde acetate - acetaldehyde is reactive and toxic.

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

1. HEXOKINASE Phosphorylation of glucose. Inhibited by its product, glucose 6-phosphate, as a response to slowing of glycolysis Not GLUCOKINASE as discussed

2. PHOSPHOFRUCTOKINASE  major regulatory enzyme, rate limiting for glycolysis  an allosteric regulatory enzyme.  measures adequacy of energy levels. Inhibitors: ATP by decreasing fructose 6-phosphate binding and citrate both indicate high energy availability Activators: ADP, AMP, low energy

AMP and ADP reverse ATP inhibition And another activator 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 CoA and fatty acids (alternative fuels for TCA cycle)  activator: fructose 1,6bisphosphate ( feed-forward)

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