1. Write the reaction for production of NAG (N-acetylglutamate).

2. Explain the MR and ML for glutamate regulation of CPS I (carbamoyl phosphate synthetase I) by NAG.

Overview of Metabolism• ATP is consumed in energy requiring

processes and is produced in energy yielding processes.

• G + Pi G6P ΔGo’ = +13.8 kJ • ATP ADP + Pi ΔGo’ = -30.5 kJ • G + ATP G6P + ADP ΔGo’= -16.7 kJ

• PEP Pyr + Pi ΔGo’ = -61.9 kJ• ADP + Pi ATP ΔGo’ = +30.5 kJ • PEP + ADP Pyr + ATP ΔGo’ = -34.4 kJ

Overview of Metabolism• The energy required for the variety of work that an

organism must do (such as muscle contraction, active transport and protein synthesis) is supplied by ATP hydrolysis (H2O omitted in above rxns).

• The amount of ATP in a cell is small and it is constantly consumed, so it must be constantly produced (otherwise, cells like heart muscle and brain/nerves die in a short time).

• The bulk of the energy for ATP production comes from “burning fuel”, the oxidation of food components (fats, carbohydrates (CH2O’s) and proteins). In this oxidation, electrons are transferred from Cs of foods to O2, so we must breathe O2 constantlyconstantly for oxidation and store fuel between meals to maintain fuel supplies.

Overview of Metabolism• The vast majority of ATP in O2 using cells is produced in

oxidative phosphorylation (OP) in a reaction catalyzed by ATP synthase (an enzyme of the inner mitochondrial membrane): ADP + Pi ATP.

• The indirect source of energy for this reaction is a sequence of redox reactions (in the electron transport chain (ET)) that result in O2 reduction: 4H+ + 4e- + O2 2H2O.

• ET produces an “energized” intermediate, which is an elevated [H+] outside the inner mitochondrial membrane that ATP synthase uses as the energy source for OP (Fig 22-29, p821).

Overview of Metabolism

• The source of the e-’s in ET is the C atoms in food molecules.

• The e- are transferred from metabolites of food to NAD+ (made from dietary niacin) and FAD (from riboflavin), which “carry” the e- to ET.

• Some e- transfer to NAD+ and FAD occurs in most metabolic pathways/cycles we’ll study, especially β- oxidation of fatty acids (FAs), but the majority occurs in the TCA cycle (ttriccarboxylic aacid).

• The main purpose of the TCA is to oxidize C: “harvest” the e- of food fuels for delivery to ET. (Fig 22-1 p 798)

Overview of Metabolism

• So the overall scheme for ATP production is to convert food molecules to the form in which they are consumed by the TCA cycle, acetyl coenzyme A (ACoA) (Fig 16-3 p 551).

• CH2O’s are polymers which are hydrolyzed to glucose (G) (+/or other sugars which are converted to G). G is converted in glycolysis to pyruvate, which is converted to ACoA in the pyruvate dehydrogenase (PDH) rxn.

• Fats (triglycerides or triacylglycerols) are hydrolyzed to fatty acids (FAs) which are converted in β-oxidation to ACoA.

• Proteins are hydrolyzed to AAs which are converted in AA oxidation to ACoA directly or via other metabolites like pyruvate.

• So the metabolism of the major food groups involves separate pathways which converge in production of ACoA, the fuel of the TCA cycle.

Overview of Metabolism• Aside from ATP production, our other main concern will be

storage of fuel after a meal and its release as needed. • AAs are stored as muscle protein and are released in short

term (> 8 hr) CH2O starvation. • FAs are stored as fat (which is the major stored fuel by far) and

released continuously since they are the main fuel for they are the main fuel for most cells most of the timemost cells most of the time.

• G is stored as the polymer glycogen and released in muscle cells to do muscle work and in liver for export to the blood to supply the brain its preferred fuel.

• G is also produced from AAs in gluconeogenesis for export to blood in times of CH2O starvation.

• Since the capacity to synthesize and store glycogen is limited, much of the G in a high CH2O meal is converted to fatty acids via glycolysis, PDH and FA synthesis.

Overview of Metabolism• The pathways and cycles mustmust work together. • For brain to function the nerve cells must get ATP

from the sequential action of glycolysis, then PDH, then TCA, then ET and OP.

• Glycogen breakdown and gluconeogenesis work in concert to increase blood G starting a few hours after a meal.

• Glycolysis does not convert G pyr at a high rate when gluconeogenesis is converting pyr G at a high rate.

• Also, a given pathway may have many functions: glycolysis is not only involved in ATP production and feeding FA synthesis, it also feeds AA synthesis and other biosynthetic pathways and the production of TCA intermediates (as distinct from ACoA).

Overview of Metabolism• So, how does a pathway “know” when to “stop” and

when to “go”? • We will focus on 2 mechanisms: • allosteric regulation of specific enzymes by effectors

that indicate the need (or lack thereof) in that cell for their activity;

• and control of activity by reversible phosphorylation in response to the hormones insulin, adrenaline and insulin, adrenaline and glucagon. glucagon.

• These hormones signal the cells, especially liver cells as to the needs of the organism.

• CO2 regulation of Hb is a molecular model for both of these mechanisms.

Purposes/Roles/Functions of Glycolysis

• 1. Produce ATP

• 2. Feed ATP production via PDH, TCA, ET, OP

• 3. Feed production of TCA intermediates

• 4. Feed FA synthesis via PDH

• 5. Feed AA synthesis via TCA intermediates

Glycolysis Net Rxn (adding all rxns)

• ATP + G G6P + ADP• G6P F6P• ATP + F6P F1,6BP + ADP• F1,6BP GAP + DHAP• DHAP GAP• 2Pi + 2NAD+ + 2GAP 2 (1,3BPG) + 2NADH• 2 (1,3BPG) + 2ADP 2 (3PG) + 2ATP• 2 (3PG) 2 (2PG)• 2 (2PG) 2 PEP • 2 PEP + 2 ADP 2pyr + 2ATP

___________________________________________• G + 2Pi + 2 ADP + 2NAD+ 2pyr + 2 ATP + 2NADH

Fates of Pyruvate

• Depending on the organism in question and the conditions under which it is operating, pyruvate may be converted to:

• Lactate

• Acetyl coenzyme A (ACoA)

• Ethanol

• Oxaloacetate

• Alanine

Gluconeogenesis• Gluconeogenesis: in the liver, pyruvate (or

oxaloacetate) is converted to G to increase [G] in the blood:

• 1. in short-term CH2O starvation: muscle protein is hydrolysed to AAs which go to the blood and to the liver where they are converted to oxac G to supply G to the brain when [G] is low in the blood.

• 2. in rapid muscle activity, lactate from muscles is converted back to G to go back to the muscles

• Reactions: 7 of the reactions are the same reactions (same Es) as in glycolysis: they just go faster in reverse during gluconeogenesis (G’neo). (Q becomes greater than K)

• The irreversible reactions of glycolysis (G’lysis) are bypassed (NOT reversed)

Net Reaction for G’neo• To bypass PK (twice/G) consumes 2ATP +

2GTP.

• Reverse of PGK (2(1,3BPG) + 2ADP 2 (3PG) + 2ATP) twice/G consumes 2ATP.

• No ATP is produced in the bypass rxns of G’neo, nor in the reversed rxns of G’lys.

• So the net rxn for G’neo is:

• 2pyr + 4ATP + 2GTP + 2NADH G + 4ADP + 2GDP + 6Pi + 2NAD+

Reciprocal RegulationReciprocal Regulationof G’lys and G’neo Enzymes

• AMP activates PFK by overriding the ATP inhibition of PFK; and AMP inhibits FBPase.

• MR for PFK: its activity feeds ATP production in glycolysis AND in OP via PDH + TCA + ET + OP

• ML: if [AMP] is high then ATP is being consumed rapidly and needs to be produced, which it will be by PFK activation

• MR for FBPase: G’neo consumes 6ATP/G• ML: if [AMP] is high then ATP is being consumed

rapidly and needs to be produced; cell can’t afford to consume ATP in G’neo when ATP production is needed, FBPase/G’neo slows in response to AMP

Hormonal Regulation of G’lys and G’neo In Liver• Glucagon and insulin exert their effects on

these pathways by regulating the “tandem enzyme” See pages 649-650 in V+V.

• PFK2 and FBPase2 activities are at 2 different active sites on each of the polypeptide chains of “the tandem E”, a homodimer (two identical subunits).

• PFK2 catalyzes: F6P + ATP F2,6BP + ADP• FBPase2 catalyzes: F2,6BP F6P + Pi • F2,6BP activates PFK (overrides PFK inhibition

by ATP)• F2,6BP inhibits FBPase• There is only one purpose (and only one effect)

of F2,6BP: to regulate PFK and FBPase.

Hormonal Regulation of G’lys and G’neo In Liver

• Glucagon is secreted in short-term CH2O starvation in response to low [G] in blood; its purpose is to cause the liver to release G into the blood by activating G’neo and glycogenolysis.

• When the tandem E is phosphorylated in response to glucagon, only its FBPase2 is active (PFK2 is NOT); this causes the [F2,6BP] to decrease.

• This removes the inhibition of FBPase by F2,6BP and G’neo goes rapidly.

• G’lys is slowed by ATP inhibition of PFK.• G’lys has to go slow (NOT consume G when [G] is

low) so as to not oppose G’neo.

Hormonal Regulation of G’lys and G’neo In Liver

• Insulin is secreted after a CH2O meal in response to high [G] in blood; its purpose is to cause the liver to store fuel by G’lys, PDH, CS, and FA synthesis, and by glycogen synthesis.

• When the tandem E is dephosphorylated in response to insulin, only its PFK2 is active (FBPase2 is NOT) ); this causes the [F2,6BP] to increase.

• PFK is activated by the [F2, 6BP] increase, and glycolysis goes rapidly to “feed” FA synthesis (The E of FA synth is also dephos, active).

• FBPase is inhibited by F2,6BP, to keep it from opposing G’lys

Hormonal Regulation of G’lys and G’neo• Liver PK is also phosphorylated in response to

glucagon. This inhibits PK to slow G’lys and keep it from working against G’neo.

• Liver PK is also dephosphorylated in response to insulin. This activates PK to work with PFK to make G’lys go to feed FA synthesis.

• In heart muscle, the tandem E has the opposite response to phosphorylation, which occurs in response to adrenalin, as compared to tandem E in liver:

• The heart muscle tandem E’s PFK2 is active (its FBPase2 is inactive), so that the [F2, 6BP] increases and PFK is activated.

• The purpose here is to activate glycolysis, so that ATP can be rapidly produced (in G’lys and in PDH, TCA, and ET + OP) so that heart muscle can have rapid activity.

Regulation of PC, Pyruvate Carboxylase • PC is the “anaplerotic” (filling up) reaction for the TCA;

it is also a G’neo rxn: pyrpyr + ATP + CO2 oxac + ADP (Oxidation of 18 of the 20 AA also produces TCA ints.)

• ACoA activates PC; PC requires that ACoA be bound to it in order to be active

• MR (for G’neo): ACoA is the product of PDH an alternative use of pyruvate (vs PC)

• ML: If [ACoA] is high, then pyr is not needed for PDH, can be “conserved as CH2O” and converted to G

• ACoA cannot be converted to G: PDH is irreversible AND THERE IS NO BYPASSNO BYPASS

• MR (for TCA): product of PC (oxac) is needed to react with ACoA, the fuel of the TCA

• ML: if [ACoA] is high, oxac is needed to react with, consume it, PC must produce oxac (overlapping MRDs for G’neo and TCA)

TCA Cycle• Purpose of TCA: oxidize C from food molecules so

that their e- can be carried to ET by NAD+ or FAD • Compare the oxidation states of C in G, lactic acid,

and CO2. The average is zero for the C in G or lactate, but it is +4 in CO2.

• Calculating oxidation states for C atoms, based on atoms or groups bonded to them:

• 1. Count -2 for =O or for –O-

• 2. Count -1 for -OX (-OH; -OR; -SR)• 3. Count +1 for -H• 4. Count nothing for ANY/ALL C• 5. sum of above counts, plus that for the C must

equal the charge shown

TCA Cycle: a Major Intersection of Metabolism

• Numerous compounds are produced from or converted to TCA ints.

• G’neo consumes TCA intermediates, as does production of some AAs

• PC produces TCA ints. (fromG), as does oxidation of 18 of the 20 AA

• FAs (and cholesterol) are made via citrate

• Failure to maintain [TCA ints.] is fatal

ET and OP are Tightly Coupled• Each of the three multienzyme complexes supplies

energy for oxidative phosphorylation to produce one ATP.

• Each of the 3 complexes acts as a “proton pump” expelling H+ from the matrix.

• The common intermediate which couples ET and oxidative phosphorylation is NOT H+, it is the H+ gradient (high [H+] outside and low [H+] in the matrix ).

Net Reactions of ET Complexes• NADHDH: 2NADH + 2H+ + 2Q 2NAD+ + 2QH2

• Cytochrome Reductase net reaction: 2QH2 + 4 cyto c (Fe3+ ) 2Q + 4H+ + 4 cyto c (Fe2+)

• Cytochrome Oxidase net reaction: 4 cyto c (Fe2+) + 4H+ + O2 4 cyto c (Fe3+ ) + 2H2O.

ATP Synthase

is a Rotary

Molecular Motor

• The a, b2, δ and α3β3 hexamer subunits are stationary, but the c barrel and the γ subunit are caused to rotate by the entry of H+ into the matrix.

• The gamma subunit is not symmetric. A different “face” of it is in contact with each of the 3 αβ pairs. Each face has different interactions with an αβ forcingforcing the 3 βs to always be in 3 different conformations.

• As gamma spins, each β goes through a sequence of conformational changes: O L T O L T etc

Binding Change Mechanism of ATP Synthase

• Properties of the 3 conformations of β:• O – “open”; low affinity for ATP/ADP: releases ATP• L – “loose”; moderately affinity for binding ADP and Pi• T – “tight”; tight binding of ATP: can’t release ATP• ATP is synthesized on the β subunit that is in the T

form. But ATP production requires that T subunit to be converted to the O form for release of ATP. If T-ATP reverts to the L form, ADP + Pi will be released.

• It is the directional sequence of conformational changes: O L T O L T etc, that causes ATP production.

Rotational Motion from H+ Flow

How OP Controls the Rates of ET and TCA• When the rate of ATP synthase is low, the H+ gradient

is consumed slowly and the gradient approaches a maximum.

• When the gradient is at max (at rest, when ATP consumption is low and [ADP] is low) then net H+ release by ET on outside is slowed by the high [H+] and H+ binding on the inside is slowed by the low [H+]: ET enzymes must “pump” H+ in order to catalyze e- transfer.

• The gradient is the product of ET; when it is high ET is slowed (and when it is low, ET goes).

• When ET is slow, the rate of NADH consumption slows, and [NADH] increases. This causes product inhibition effects that slow TCA. And ET produces NAD+ slowly, [NAD+] decreases, and enzymes of TCA have a lower [S] supply, slowing them.

ATP Produced G’lys, PDH, TCA, ET, and OP• G’lys, PDH, and TCA • Converted G to 6 CO2

• Produced 2 ATP + 2 GTP• Produced 2 FADH2 + 10 NADH• When ET consumes the 2 FADH2, OP

produces 4 ATP• When ET consumes the 10 NADH, OP

produces 30 ATP, for a net of 38 ATP / G• When the glyerol-3-P shuttle is used:• 4 FADH2 go into ET, and 8 ATP from OP, and• 8 NADH go into ET, and 24 ATP from OP. This

nets 36 ATP / G

Glycogen Metabolism • Purpose: Glycogen is

a branched polymer of glucose; it is the stored form of G.

• The many branches each have a C#4 end at which GP and GS can act for rapid response.

• Glycogen is stored after a meal for release:

• From liver when blood [G] is low to supply brain; OR

• In muscle for rapid activity.

FAs, Fatty Acids• FAs are a much more efficient form of stored

fuel: 9kcal/g (9 Cal/g) vs. (4 Cal/gG); also glycogen binds two times its weight of H2O. A typical man would have to store ~ 90 kg of glycogen (~200lbs) if he was to have the same energy as in the ~15 kg fat he stores.

• Although glycolysis is a major fuel consuming pathway, FAs are the main fuel (except in brain, RBCs, rapid muscle activity).

• Because of the above, glycogen storage is limited and “xs G” is converted to fat via glycolysis, PDH, CS and FA synthesis.

FA Use as Fuel• FAs are released from storage (as triacylglycerol) by

the hydrolytic action of hormone-sensitive lipase, which is activated by phosphorylation by PrK in response to adrenalin or glucagon, deactivated by dephosphorylation by PP1 in response to insulin.

• FA’s then travel from adipose cells (“cytosol” is mainly a fat globule) via blood to cells that use them, where:

• FA’s are prepared in the cytosol (cytoplasm) for transport to the mitochondrial matrix, where they are converted to ACoA in oxidation.

• FA activation: (costs 2 ATP)• a) FA + ATP PPi + FA – AMP; • b) FA – AMP + CoASH AMP + FA – SCoA

Ketone Body Production

• The moderate rate of production of acetoacetate, hydroxybutyrate and acetone that occurs normally in the liver mitochondrial matrix delivers “water soluble FA fragments” to other cells via blood for use as fuel.

• Since this process involves unregulated enzymes, the buildup of ACoA in diabetes overproduces these compounds to toxic levels.

FA Synthesis

• FA synthesis is a liver pathway

• The net effect is to build up the CH2 chain by joining ACoAs’ acetyl gps. and reducing (and hydrogenating) the C=O of ACoA.

• The ACoAs for FA synthesis don’t come from -oxidation. Rather it’s the “xs G” that enters liver cells after a meal and goes through insulin stimulated glycolysis and PDH.

Amino Acid (AA) oxidation • Introduction• 1. Part of the C’s of some of the AAs are convertible to

ACoA, either directly or via acetoacetate or pyr. (and less directly, so are the others via TCA int oxac PEP pyr ACoA.) (These AAs are “ketogenic)

• So, these C’s of xs AA intake (in relation to need for protein synth) are used as fuel, just like dietary CH2O’s, fats.

• 2. Part (or all) of the C’s of 18 of the AAs can be converted to TCA intermediatesconverted to TCA intermediates, which can be converted to G (TCA int oxac PEP G). These are referred to as the “glucogenic” AAs.

• AAs from digestion of muscle protein are the main source of C for gluconeogenesis in CH2O starvation

Transaminations (trnsams)• Each AA can be converted to the corresponding

keto acid by at least one transaminase. This AA is oxidized in this rxn, but kg is reduced to glutamate at the same time so there’s not a net AA oxidation here.

• The amino group transferred to kg (---> glu) is toxic when released as NH3, this ammonia is detoxified by conversion to urea in the urea cycle (NH3 can be excreted). Net oxidation occurs by coupling of trnsam with glutamate dehydrogenase (GDH).

• GDH: glu + NAD+ kg + NADH + NH3 • This rxn running in reverse when [NH3] is very high

depletes TCA ints, interferes with TCA +ET + OP in brain cells and causes the delirium/dementia in liver damaged patients.