number 7 Done by حسام أبو عوض Corrected by Shahd Alqudah Doctor Nafeth Abu Tarboush 1 P a g e
As we have studied before, in the fourth reaction of the Krebs cycle, α- ketoglutarate is converted into Succinyl-CoA using an enzyme called α- ketoglutarate dehydrogenase. Yet it is unclear how NAD + has been reduced into NADH although no change has happened on the number of Hydrogen atoms in α-ketoglutarate when it was converted to succinyl CoA, this will be explained later on. In reality, α-ketoglutarate dehydrogenase is not a single enzyme it is a complex [in a complex more than one enzyme work together in which the product of the first enzyme would be the substrate of the next one. Be careful, NOT a single enzyme with several subunits(same function), but a complex (many enzymes and different functions)]. This means that this enzyme complex performs more than one function (which is logical), and these functions are: 1- Decarboxylation 2- Transacetylation ( transport of acyl group) and coupling of CoA 3- Oxidation Each of these functions is carried by an enzyme, so we have three enzymes in this complex: Enzyme #1 It is a decarboxylase. The coenzyme it needs to function is TPP [Thiamine pyrophosphate, the active form of thiamine (B1) (refer to summer course)]. TPP attacks the bond between the carbonyl group (C=O) and the terminal carboxyl group (COO ) weakening it and so the carboxyl group leaves the molecule and exits as CO₂ Now the carbonyl (C=O) group binds to TPP and so our molecule becomes bound to the enzyme. (Extra info: the C=O gets reduced to C-OH bond after getting attached to TPP) As we know, the enzyme doesn t change during the reaction; so our enzyme has to return to its original form (without acyl group). Here comes the role of enzyme #2. Enzyme #2 (Transacetylase ) CoA is added to the molecule here. 2 P a g e
The coenzyme here is called Lipoic Acid which has two sulphur atoms bound to each other covalently (a disulphide bridge). One of these sulphur atoms binds to our molecule and in the process our molecule becomes unbound to TPP and enzyme #1 returns to its original form. This process results in the break-down of the disulphide bridge between the two sulphur atoms, the other sulphur atom (not bound to our molecule) picks -up a hydrogen atom from the solution forming a thiol group (SH). Then CoA binds to our molecule and our molecule is released in its final form (no more processing occurs to the molecule after this, Succinyl CoA has already been formed now and leaves the complex at the level of enzyme 2) Now the sulphur atom that was bound to our molecule also picks up a hydrogen atom from the surrounding solution and another thiol group forms. Now to return our enzyme to its original shape we need the aid of enzyme #3. Enzyme #3 This enzyme is a dehydrogenase. Its coenzyme is FAD; NAD+ is changed to NADH here. FAD picks up the two hydrogen atoms from the thiol groups in enzyme #2 and the disulphide bridge reforms returning enzyme #2 to its original form. Then, for this enzyme #3 to return to its original shape, FADH₂ passes on its hydrogens to NAD+ forming NADH and H+. Notice that NAD+ can't take the two hydrogen atoms lost from the thiol groups, so the hydrogens are transferred to FAD first then to NAD+ (hydride ion). 3 P a g e
Important notes: The mechanism described above also applies to the Pyruvate dehydrogenase complex (changes pyruvate to acetyl CoA) and to the branched chain α-keto-acid dehydrogenase complex (changes ketoacids to acetyl CoA in the metabolism of amino acids). A deficiency in vitamin B1 (thiamine) means that enzyme #1 in the complex cannot work properly and so free substrates concentrations (αketoglutarate, branched chain α-keto-acids and pyruvate) increase in blood. Arsenic is a lethal poison that attacks the energy metabolism process (also cyanide attacks energy metabolism and most lethal toxins) by binding to the two sulphurs of enzyme #2 not allowing it to function and so causing the entire energy production mechanism to fail (this happens in pyruvate dehydrogenase complex as well). This enzyme complex contains five coenzymes that act as carriers or oxidants for the intermediates of the reactions. E1 requires TPP, E2 requires lipoic acid and CoA, and E3 requires FAD and NAD +. Note: TPP, lipoic acid and FAD are tightly bound to the enzymes and function as coenzymes-prosthetic groups, but CoA & NAD + come from the solution and they are not bound to the complex. Bioenergetics of Krebscycle Efficacy is the ratio of the amount of actual product we get (energy in our case) to the calculated (theoretical) amount we are supposed to get. Efficacy= (Actual amount / Theoretical Amount) * 100% To calculate the theoretical amount of energy for Krebs cycle: we find the energy produced by acetate (the molecule from which we extract energy, other molecules all return to the cycle) by burning one mole of acetate completely in a calorimeter to produce CO₂ and H₂O. This gives us a value of 228 kcal/mol. The amount of energy we actually get is 207 kcal/mol. This gives us an efficiency of 90.8% ((207/228) *100%), and this is way higher than any machine humans have ever produced. 4 P a g e
Krebs Cycle Regulations As we have studied in the summer course, metabolic pathways are always regulated, and the Krebs cycle is not an exception. We said that in any pathway it is important to have a high level of regulation on the first step and the rate limiting step. Our first step is the production of citrate via the enzyme citrate synthase. If too much citrate (molecules only act as regulators when their concentrations are high) is present, citrate itself inhibits citrate synthase by competing with oxaloacetate. NADH and succinyl-coa also play a role in regulating this enzyme. Our rate determining step is the production of α-ketoglutarate from isocitrate. If ADP levels increase (even if just a bit) the activity of isocitrate dehydrogenase enzyme increases a lot. Also,Ca++ activates isocitrate dehydrogenase (Ca++ increases during muscle contraction so there is a need of more energy activation). NADH levels, however, inhibit the action of isocitrate dehydrogenase when they increase. Most enzymes that have to do with energy production (α-ketoglutarate dehydrogenase, isocitrate dehydrogenase, pyruvate dehydrogenase, etc...) are activated by Calcium. Also, it is extremely important to monitor the concentrations of the main products of the cycle and regulate its reactions accordingly and therefore several enzymes are regulated by the concentrations of NADH and NAD and the concentrations of ATP and ADP. Why isn't FADH2 a regulator of the cycle? Because it is always bound to a protein and it isn't free to activate or inhibit enzymes other than the one it is bound to. 5 P a g e
Regulation of Citric Acid Cycle depends on two major concepts: 1. NADH/NAD+ ratio: More NAD+ >>> cycle will be activated More NADH >>> Cycle will be inhibited The enzymes that are affected by this regulation method are the ones that produce NADH, which are isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase. NADH will inhibit these enzymes by feedback inhibition. 2. ATP/ADP ratio : More ATP >>> cycle will be inhibited. More ADP >>> cycle will be activated. One enzyme is affected by this method, which is the isocitrate dehydrogenase (the rate-limiting enzyme of the cycle) 6 P a g e
The intermediates The intermediates produced in the Krebs cycle might be, at any point, in excess of our need. In such a scenario they can simply leave the mitochondria and participate in other reactions. 1. Citrate: It can inhibit the rate determining step in glycolysis (in the cytosol) by inhibiting phosphofructokinase enzyme (Pyruvate Acetyl CoA ). It can also participate in fatty acids synthesis alongside acetyl CoA. 2. α-ketoglutarate: It can participate in amino acid synthesis by being changed from a keto-acid to an amino acid with the help of Pyridoxal Phosphate (PLP) (which is made from vitamin B6 - Pyridoxine). α- Ketoglutarate can also be used in the production of GABA (ϒaminobutyric acid) which is a neurotransmitter, by the synthesis of glutamate (a-ketoglutarate glutamate GABA) 3. Malate: It can be used in gluconeogenesis (production of new glucose from from noncarbohydrate precursors). 4. Succinyl CoA: It can be used in the production of heme (succinyl CoA changed to propionyl CoA). 5. Oxaloacetate: amino acid synthesis (aspartic acid). Keep in mind the conversion of the following keto acids to amino acids by transamination reaction: Oxaloacetate Aspartate Pyruvate Alanine α-ketoglutarate Glutamate 7 P a g e
8 P a g e Anaplerotic Reactions These are reactions that replenish (form more of) the intermediates of the Krebs cycle. Acetyl CoA: Carbohydrates, fatty acids and amino acids all can be used to produce it. α-ketoglutarate: Glutamate can be converted to α-ketoglutarate. Succinyl CoA:Propionyl CoA can be converted to succinyl CoA. Propionyl CoA can be made from valine, isoleucine and odd chain fatty acids. Fumarate: Can be produced from amino acids, and urea cycle. Oxaloacetate: Can be produced from aspartate and pyruvate. The conversion of pyruvate to oxaloacetate is the main anaplerotic reaction. This reaction is catalysed by the enzyme pyruvate carboxylase (adds a COO ). This enzyme is activated when the concentration of acetyl CoA is high [as to produce more of the other reactant; acetyl CoA needs to react withoxaloacetate]. This enzyme s coenzyme is biocytin (Biotin + Lysine, biotin is vitamin B7). Pyruvate carboxylase is found in high concentrations in the liver and the kidneys because there is too much gluconeogenesis there (in which malate is used up), so another source for production of oxaloacetate is needed. gluconeogenesis >> malate >> oxaloacetate Oxidative Phosphorylation It is the fourth and last stage of energy production. The electron carrying molecules (NADH and FADH₂) produced by the Krebs Cycle will be used in this process. However, movement of electrons does not generate ATP at all, that is why the electron transport chain is just part of the oxidative phosphorylation, if we are to divide this name (oxidative phosphorylation) into two parts then the electron transport chain would only refer to the first part, oxidative, because the electron transport chain s final electron acceptor is oxygen.
Looking closely at the electron transport chain we notice that the molecule that loses the electrons gets oxidized while the one that gains them gets reduced, so it is also correct to describe the electron transport chain as a series of redox reactions. As the name suggests, the second part of the process, phosphorylation, is always coupled to the first part, oxidative. The outer-membrane of mitochondria is permeable to anything that weighs less than 5000 Daltons, while the inner-membrane is not permeable to anything even to H+ (which is just a proton -the smallest ion-). Therefore, anything to pass through the inner-membrane must do so through channels. The exact mechanism of oxidative phosphorylation was discovered in 1961 by Peter Mitchell where he divided the process into 3 main concepts: 1- Electrons move through a chain of membranebound carriers (prosthetic groups) according to the potential difference; so when they move, there is always some energy released in each reaction till reaching the final electron acceptor. 2- This energy is used to pump H+ ions from the matrix to the intermembranous space of the mitochondria(through channels that allow H+ ions to move out against the gradient, but not back in), thus creating a high electrochemical gradient in that intermembranous space (H+ ions cannot come back through normal diffusion, they need a channel). 3- The only channel that allows H+ ions to move back into the matrix is ATP-synthase, which, in the process of moving H+ ions back, produces ATP. If there was no channel for H+ to return back to the inside of mitochondria, these ions would make a pressure on the IMM (positive charges), and by electrons' movement this pressure would increase until it stops the H+ pumping from inside to intermembranous space so the electron transport chain would stop. 9 P a g e