Pyruvate and the Krebs Cycle

How is PDH regulated?

-Acetyl CoA and NADH can be made in mitochondria from a number of sources. If they build up, they force the PDH reaction backwards until acetylated E2 is made and the enzyme is blocked -prevents carbohydrate carbons from entering the mitochondrial metabolic pathways and further clogging up the system. -in other words, Acetyl-CoA and NADH when plentiful inhibit forward reaction of PDH -AMP and ATP are allosteric regulators that: 1. allow PDH to work when ATP is needed (AMP is present) 2. inhibits PDH when ATP is plentiful

What enzymes activate and inactivate pyruvate dehydrogenase? How does this work?

-Because this complex serves as a gateway for a major flow of carbon into the mitochondrion, it is additionally subject to regulation by phosphorylation to further clamp off carbon inflow if the mitochondrion becomes too congested -same compounds that work to make acetylated E2 activate the kinase -Kinase converts the enzyme to an INACTIVE form a. Kinase activated by NADH, acetyl-CoA b. ATP-dependent (adds a phosphate to PDH to inactivate it) -Phosphatase converts the enzyme to an ACTIVE form a. Phosphatase activated by Magnesium and Calcium b. Insulin also activates phosphatase c. enzyme uses water to break off the phosphate to activate PDH

What is all used and produced in the krebs cycle?

-For every two acetyl carbons that enter the cycle, two CO2 leave in oxidative decarboxylation reactions; hence, in our bodies, one CANNOT convert even chain Fatty Acids, degraded to acetyl Co A, to sugar -The cycle carries out 4 oxidation reactions producing 3 NADH and 1 FADH2 which serve as electron transfer chain fuel -the cycle actually produces 1 NTP, either ATP or GTP, depending on the tissue (succinyl CoA synthetase) -the cycle also is involved in biosynthetic functions and in gluconeogenesis

how is mitochondrial isocitrate dehydrogenase regulated?

-a major metabolic fork in the road in the mitochondrion. -ADP is a significant POSITIVE allosteric regulator. As ADP increases, IDH activity increases and more of the citrate/isocitrate can go around the cycle to generate reduce electron carriers for ATP generation -If ATP/ADP ratio is high, energy is available and IDH is turned DOWN. Citrate instead goes into cytosol where it can be cleaved to acetyl CoA for biosynthetic purposes. -for all the dehydrogenases, activity is pegged to respiration state. If reduced electron carriers accumulate because of decreased availability of O2 or inhibition of the electron transfer chain, there will not be enough NAD/FAD to run the cycle and it will slow down. (NADH will INHIBIT isocitrate dehydrogenase)

describe the structure of coenzyme A

-abbreviated CoASH showing emphasis is on the reactive thiol -in fatty acid synthase, a derivative of CoA without the AMP (4-phosphopantothenyl group) is attached covalently to the protein and acts as an acyl carrier

Describe the role for succinyl CoA in metabolism

-amino acid (Ile, Val, Met) degradation breaks down to succinyl CoA -Krebs cycle uses succinyl CoA -odd or branched chain fatty acid oxidation use succinyl CoA -succinyl CoA is used in GTP synthesis -Succinyl CoA is used in heme synthesis (8 succinyl CoAs per heme) -in cells that use ketone bodies as fuel, acetoacetate must be converted first to acetoacetyl CoA. Succinyl CoA is the CoASH donor -any time one removes a KC intermediate for another purpose, one has, in effect linearized the cycle. To continue to burn fuel, one must synthesize de novo a new molecule of OAA to replace the intermediate that was removed

Explain how amino acid carbon skeletons are oxidized using the citric acid cycle

-assume a Glu enters TCA cycle at alpha-ketoglutarate -it goes around the cycle til it hits OAA. 4 carbons are still left -OAA is not fuel for the cycle -take all but one molecule of the OAA, convert it to PEP (PEPCK), convert the PEP to pyruvate (pyruvate kinase) and the py ruvate to acetyl CoA -now run the acetyl CoA through the cycle as fuel

How does aconitase play a part in iron regulation?

-cytoplasmic aconitase is the iron response element binding protein that controls iron sequestration and dispersal in the body. Its activity in this role depends on the ability of the Fe/S center to form when Fe is abundant and disassemble when Fe is scarce -In high iron environment, aconitase (cytoplasmic iron response element binding protein) stabilizes with iron which activates Ferritin which stores iron and decreases expression of transferrin which brings iron into the cell -in low iron environment, aconitase is unstable and does not bind to transferrin, which stabilizes it and brings iron into the cell. Ferritin expression is decreased.

describe the conversion of alpha-ketoglutarate to succinyl CoA

-enzyme: alpha-ketoglutarate dehydrogenase complex -succinyl CoA contains a high energy thioester bond which will be used to generate NTP synthesis in the next step -oxidative decarboxylation of an alpha-ketoacid, just like PDH. Chemistry and cofactor requirements (lipoate, NAD, FAD, TPP) the same but specificity of E1 and E2 are different since a different substrate is involved

Explain oxidation of pyruvate to acetyl coA

-enzyme: pyruvate dehydrogenase complex (E1 + E2 + E3) -cofactors: CoA-SH, NAD, TPP, lipoate, FAD -oxidative decarboxylation of an alpha-ketoacid -all oxidative decarboxylations work by the same chemistry, but substrate specificity is different between complexes -always use the same complement of co-factors: NAD, FAD, thiamine-PP, Coenzyme A, and lipoic acid. All are derivatives of vitamins

What is Beriberi?

-extreme thiamine deficiency disease -extreme weakness (Sinhalese) -also often accompanied by symptoms of Wernicke's encephalopathy -dry beriberi - extreme fatigue and muscle atrophy but no edema -wet beriberi - extreme fatigue along with edema due to congestive heart failure and capillary damage with decreased vascular resistance. A high output cardiac failure. Very high systolic blood pressure accompanied by very low diastolic blood pressure -in third world countries, related to vitamin-deficient diets based on white rice with little other vitamin sources - in US, generally related to alcohol abuse leading to extreme thiamine deficiency from inhibition of thiamine uptake in the gut

Describe how NADPH is generated using malic enzyme

-liver specific (mostly) -cytosolic -Reaction: malate + NADP -> Pyruvate + CO2 + NADPH + H+ -oxidative decarboxylation -in liver, adds to ability of pentose phosphate shunt to make NADPH -not essential but makes NADPH-utilizing reactions go more efficiently

How does the non-heme iron center in aconitase function?

-non-heme iron centers reversibly form depending on Fe availability. This Fe4S4 center contains S-, which in acid pH, comes off as H2S -in redox reactions, these centers typically can hold one electron -insufficient Fe availability will have a direct negative effect on the KC -the cytosolic form of aconitase acts as an iron sensor, binding to the Iron Response Element and controlling production of ferritin (depressed in times of low iron) and transferrin receptor (increased in times of low iron)

How does arsenite affect pyruvate dehydrogenase complex?

-reduced form of lipoic acid will react with arsenite (AsO2-) to form an As bridge between the two sulfurs. This covalently blocks E2 and hence kills the whole complex -covalently modifies and inactivates the enzyme

Describe the regulation of alpha-ketoglutarate dehydrogenase complex

-regulation in terms of excess NADH and succinyl CoA backing up the enzyme to give an acylated E2 is just like in PDH. -a difference, is that this is not a gateway enzyme. It does not control a major flux of substrate into the mitochondrion. Therefore, unlike PDH, it is not regulated by reversiblep hosphorylation/dephosphorylation

What is lactic acidosis?

-serum lactate is normally <2 mM. If elevated lactate is prolonged, look for metabolic or physiologic abnormality -inhibition of PDH or other part of aerobic pathways in mitochondria causes switch to anaerobic glycolysis and increased lactate production

What is pesticide 1080 and how does it affect the krebs cycle?

-sodium monofluoroacetate -converted by citrate synthase to monofluoroisocitrate which is a very strong noncovalent inhibitor of aconitase -large scale spraying in New Zealand to control the Australian Possum population which is devastating NZ forests -potential for large public health problem due to exposure

Describe the structure and use of lipoic acid

-the attachment of the carboxy of lipoic acid covalently to a side chain amino of the E2 lysine forms an amide bond. Hence, lipoic acid attached to a protein is more properly called lipoamide -reduced form of lipoic acid will react with arsenite (AsO2-) to form an As bridge between the two sulfurs. This covalently blocks E2 and hence kills the whole complex

Explain the mechanism of pyruvate dehydrogenase

1. E1 does two things. It uses TPP to decarboxylate the pyruvate and then catalyzes the attack of the resulting 2C unit on the lipoic acid residue 2. oxidation of the keto carbon of pyruvate occurs when the C2 carbanion attached to TPP attacks and opens the oxidized lipoic acid disulfide forming an enzyme bound thioester 3. CoASH attack on the acetyl lipoic acid is simply a thioester transfer to produce acetyl CoA and open up the enzyme 4. E3 with its enzyme-bound flavin, removes electrons from the lipoic acid and transfers them to NAD to make NADH which leaves the enzyme

Where is energy produced in the citric acid cycle?

1. NADH produced during isocitrate dehydrogenase 2. NADH produced during alpha-ketoglutarate dehydrogenase 3. GTP (ATP) produced during succinyl-CoA synthetase 4. FADH2 produced during succinate dehydrogenase 5. NADH produced during malate dehydrogenase

What are the "rules" of metabolic cycles?

1. there is a fuel consumed in the cycle 2. there is a carrier which brings the fuel into the cycle and must be regenerated for the cycle to continue

What is the major fuel for the Krebs Cycle?

Acetyl CoA

What funnels to Acetyl CoA? What is it used for?

Breakdown of amino acids, fatty acids, and sugars all generate acetyl CoA and reduced electron carriers in the form of NADH or FADH2 which are used in mitochondria to generate energy for ATP synthesis

describe pyruvate dehydrogenase dficiency

E1 and E2 are substrate and therefore complex specific. However, E3 is the same for all alpha-ketoacid oxidative decarboxylase complexes. A deficiency in any of the subunits will cause a PH deficiency -classic PDH deficiency, and the most severe because it affects all complexes, is caused by an E3 mutation. It's called Leigh's Disease and produces pinpoint focal necrotic lesions in the brain. Either fatal in infancy or produces a severely compromised state of life.

What is the fuel and what is the carrier for the citric acid (krebs) cycle?

acetyl CoA is the fuel Oxaloacetate (OAA) is the carrier

Describe the conversion of citrate to isocitrate

enzyme: aconitase -a reversible dehydration/rehydration in which the water is put back on aconitase in the opposite orientation -by itself, the enzyme actually prefers citrate synthesis but in the cell, it is pulled to the right by the irreversible steps of the krebs cycle -the enzyme requires a non-heme iron sulfur cluster to orient the substrate on the active site. One of the few times where such a structure is not used in a redox reaction as an electron carrier -movement of the OH places in a position alpha to one CO2 and beta to the other. This ultimately allows creation of alpha and beta keto acids needed to destabilize the CO2 groups for decarboxylation

Describe the synthesis of citrate in the krebs cycle

enzyme: citrate synthase -by itself, a highly exothermic reaction. However, in the cell, it basically balances the highly energetically unfavorable malic dehydrogenase step that precedes it -the citrate made here also serves as a carrier of acetyl CoA to the cytosol for use in fatty acid synthesis or other acetylation reactions -Thioester of acetyl CoA acts as an electron withdrawing group to crete a carbanion on the methyl carbon. This attacks the ketone of OAA forming a C-C bond - citryl CoA. The CoASH is then eliminated to drive the reaction forward.

describe the conversion of fumarate to L-malate

enzyme: fumarase -as an isolated enzyme, it carries out a simple reversible hydration of the double bond of fumarate. Hence, could also be called fumarate hydratase -in our cells, the reaction is driven forward because it is coupled to a series of biologically irreversible steps

Describe the conversion of isocitrate to alpha-ketoglutarate

enzyme: isocitrate dehydrogenase -another example of a beta-hydroxyacid oxidative decarboxylation. Like that in the conversion of 6-phosphogluconate to ribulose-5-P in the PP shunt. The hydroxyacid is oxidized to a ketone, forming a beta-ketoacid which readily decarboxylates -the mitochondrial form uses NAD as an electron acceptor. It also requires ADP as an allosteric activator. Thus, at high ATP/ADP ratios, the enzyme activity decreases and citrate can go to the cytosol as an acetyl donor for biosynthesis. -alpha-ketoglutarate is a major intersection point in metabolism Amino acids with 5 carbons in a row are degraded to it, and it serves as a biosynthetic starting point to make these amino acids

Describe the conversion of L-malate to oxaloacetate

enzyme: malate dehydrogenase -regeneration of the original KC carrier by a NAD-dependent enzyme. In the direction shown, the delta G for this reaction alone is very positive. However, in the cell this positive delta G is balanced by the negative DG of the citrate synthase reaction -this enzyme plays an important role in the malate aspartate shuttle which allows electrons from cytosolic NADH to enter the mitochondrion. It is also important in allowing KC intermediates to be ultimately converted to OAA in the cytosol for use in gluconeogenesis

describe the conversion of succinate to fumarate

enzyme: succinate dehydrogenase -only KC enzyme that is membrane bound. Embedded in the inner mitochondrial membrane. Binds to another protein called ET of electron transfer factor. Together this complex makes up Complex II of the ETC -in the reaction, electrons are removed from succinate and transferred to the enzyme bound FAD. ETF then takes the electrons and transfers them to a CoQ to make CoQ red. This is the fuel for Complex III of the ETC. -malonate is a strong competitive inhibitor of this enzyme

Describe the conversion of succinyl CoA to succinate

enzyme: succinyl-CoA synthetase -energy to thioester bond of succinyl CoA is used to drive the synthesis of GTP or ATP depending on the tissue involved -ATP made an go to cytosol via ATP transporter. GTP that is made transfers its P to ADP to make ATP via the action of nucleoside diphosphate (NDP) kinase. GTP needed for cytosolic reactions is generated in the cytosol via the action of NDP kinase -by itself, this reaction is reversible. However, it is pulled to the right by the irreversible reactions of the cycle

describe dry beriberi

gradual degeneration of the long nerves of the legs then the arms lead to skeletal muscle atrophy -damage probably due to inability to utilize glucose aerobically (PDH deficiency) leading to depressed ATP production, accumulation of lactate, and local acidification of tissues

describe acetyl CoA

thioester thermodynamically unstabe Delta G: about -8kcal/mole to produce from pyruvate through PDH