Chapter 16 Citric Acid Cycle

Transcription

1 Chapter 16 Citric Acid Cycle Now will get the rest of the E out of pyruvate by completely oxidizing to CO 2 and H2O Aerobic phase called respiration More narrow definition, cellular respiration to differentiate from organismal breathing Actually part of a 3 step process (figure 16-1) Step 1 last chapter Step 2 chemical conversion of pyruvate to CO 2 but H transferred to carrier NADH and FADH doesn t generate much E, only a single GTP 2 Step 3 NADH and FADH transfer H to O to make H2O. Here is where we get the bulk of the E, and lots of ATP via oxidative phosphorylation. Chapter 19 Since early conditions on earth didn t have O 2 this is thought to have evolved later Main bulk of reaction will look at called the citric acid cycle or the Kreb s cycle after its discoverer. Several places to feed other compounds into this cycle, and several places to siphon off product for synthesis of other things, so has a central role in metabolism, and has complex control (you thought glycolysis was tough) 16.1 Production of acetate Several different ways compounds can enter the citric acid cycle. Many AA, pyruvate from glycolysis, and fatty acids enter as an acetate groups attached to coenzyme A While this is performed as a single enzymatic reaction, is very complex multi enzyme complex called pyruvate dehydrogenase complex (PDH) and contains 5 different coenzymes derived from vitamins and needs a bit of an explanation same complex structure is also used by two other enzymes system, so if you get this down you will understand the others Also note the location. We have now shifted to an enzyme complex in the mitochondria. Glycolysis was done by complexes in the cytosol, and at this point the pyruvate needs to move into the mitochondria, and all subsequent reaction we will talk about take place in the mitochondria

2 2 A. Oxidization of pyruvate to acetyl-coa and CO 2 overall reaction takes place in pyruvate dehydrogenase complex reaction called oxidative decarboxylation ÄG = kj/mole Very large and irreversible + note in this reaction 2 electrons and an H are given to NAD as a hydride - (H ) in the mitochondria the NADH. This NADH is then used to generate about 2.5 ATP through oxidative phopshorylation (chapter 19) B. Pyruvate Dehydrogense Complex - 5 coenzymes the above reaction involved 3 different enzymes and 5 different coenzymes (NAD, FAD, TPP, Lipoate and coenzyme A (Just look at structures now, will talk about mech next page) NAD (nicotinamide adenine dinucleotide) derived from vitamin niacin or nicotinic acid. An electron carrier that works in 2 electron steps. Had in chapter 14 skip FAD (flavin adenine dinucleotide) derived from riboflavin (Vitamin B 2), another electron carrier, but can work in 1 electron steps Structure Look at here, but will give more details in a minute TPP (thiamine pyrophosphate) derved from thiamine (vitamins B 1) used to manipulate chemistry adjacent to carbonyl groups Structure and typical mech figure (look at details now) CoA (coenzyme A) derived from pantothenic acid, used as a soluble acetate group carrier that can travel from one enzyme to the next in solution. Acyl group attached to Co A is high energy intermediate and is readily donated to other groups Structure 16-3 (look at details now) Lipoic acid (can be synthesized in you body so not a vitamin) used as an acetate carrier that is anchor in a complex undergoes reversible thiol oxidation and reduction, much like cystine in a protein. Structure 16-4 (Look at now)

3 3 C. Complex contains 3 enzymes pyruvate dehydrogenase (E 1) Dihydrolipoyl transacetylase (E 2) Dihydrolipoyl dehydrogenase (E 3) Many copies of each, exact composition varies in each organism Bovine kidney structure (Figure 16-5) About 50 nm diameter. 5x larger than entire ribosome! Core cluster 60 copies of E 2 3 functional domains Lipoyl domain - contans 2 lipoic Acids E 1 E 3 E1-E 3 binding domain Acyl transferase domain Domains separate linker of rich in Ala, Pro,+/- to keep domains separate Each with a TPP Each with a bound FAD Also two regulatory proteins A protein kinase A phosphoprotein phosphatase Basic structure conserved during evoluion D. Reaction Mech involves substrate channeling overall reaction mech shown in figure 16-6 step 1 pyruvate bound to TPP on E 1 and is decarboxylated See TPP reaction mech figure for more details step 2 the Lipoic acid (oxidized) on E 2 get reduced and removes the acetate product to regenerate E 1 This lipoic acid is attached to a lysine (figure 16-4) This makes it a long attachment that can move Note: this motion is shown in figure step 3 free CoA comes in, and acetate transferred to CoA which can leave step 4 lipoic acid still reduced, so transfers electron to E 3 bound FAD so it is oxidized agin and FAD reduced to FADH 2 + step 5 NAD comes in and takes electron from FADH 2 so enzyme is regenerated NADH is released to find its fate in the mitochondria Note how this reaction mechanism is really streamlined by use of lipoic acid to move things around. Substrates don t have to diffuse one and off, they are there ready to react. An example of substrate channeling

4 Mutations in any protein, or vitamin deficiencies can really screw things up Beri-Beri disease results from deficiency in Thiamine, characterized by loss of neural functions -originally seen in populations living on polished rice (polishing the rice removed the thiamine) - fish project linking lack of thiamine to fish reproduction in lake Oahe Can also be seen in alcoholics because getting calories without vitamins 16.x FMN & FAD(actually chapter & 536) FMN Flavin mononucleotide FAD Flavin adeninedinucleotide Proteins that use either of the F s are called flavoproteins Figure page 536 another electron carrier another vitamin - riboflavin (B 2) There doesn t seem to be much information for disease state due to lack of riboflavin Differences from NAD(P) 1. Usually very tightly bound to enzyme so doe NOT diffuse around cell (Sometimes covalently attached) As shown in figure can take one or two H + e So seen in a wider range of reactions To differentiate between state FMN, FMNH, FMNH 2 FAD, FADH, FADH 2 Also have shift in absorbance Oxidized max about 570, reduced max around 450 o Since tightly bound to protein, protein has great influence of E. So it varies from protein to protein. Flavoproteins tend to be very complex, often other thing involved in e transfers including other F s, inorganic ions (often Fe or Mo) 4

5 The Citric Acid Cycle first example of a cycle Figure 16-7 start by adding acetate from acetyl CoA to 4C oxaloacetate to get citric acid (Hence name of cycle) Then thru series of steps 2 CO are removed (NOT the C rom the acetate!) 2 And 3 NADH, 1 FADH 2 and 1 GTP generated and oxalacetate regenerated to begin cycle again Cycle is nice 1 oxaloacetate can oxidized infinite # of acetate 4, 5, & 6C intermediates serve both and sources for synthesis of other compounds, and a ways to bring in other compound for oxidation In eukaryote entire cycle and the subsequent oxidative phosphorylation take place in mitochondria in prokaryote take place in cytosol with oxidative phosphorylation taking place in plasma membrane A. Chemical Sense of the Citric Acid Cycle First of all the Acetyl CoA that we are oxidizing in the citric Acid cycle does not come only from pyruvate, but it can come for fatty acid oxidation or other carbohydrates or proteins, so we have many oxidative pathways focused down to this cycle Figure 15-1 Second of all, since we have lots of different pathways focused on this one method of oxidation, we want it to be as efficient as possible so we get the most energy we can out of the acetate group. If oxidize the acetate group in the simplest manner we simply complete the oxidation of the COOH group and go to methane (CH 4) and CO 2. The problem with this is that methane is very stable, and only a few methanotropic bacteria have the enzymes needed to break it down. So this would throw away lots of our potential energy. Instead we need to work on that CH 3 group and get as much of its energy as we can. Back in chapter 13 we talked a bit about the chemical logic, and one of the thing we saw was that methyl groups are more reactive when the are directly attached to a COO group or 1 methyl group away from a COO group. Thus what the Citric Acid cycle is going to to is to take that CH 3 group and put it adjacent to other groups to it becomes more reactive, and we will use step by step reactions to slowing oxidize it down by small steps to get the most energy was can out of it. Watch as we do different reactions to put different functional groups beside that CH so we can break its bonds efficiently 3

6 6 B. Citric Acid Cycle (8 steps) 1. Formation of citrate Citrate synthase 0 ÄG ' = kj/mol citroyl-coa is seen as a transient intermediate in the enzyme s active site See Mech figure 16-9 [oxaloacetate] is normally very low thus, if this reaction didn t have such a large, favorable ÄG might go in reverse So large favorable ÄG necessary start to the cycle Co-A liberated and can go back and get another acetate is an ordered reaction (oxaloacetate must bind first then acetyl CoA) --doesn t have a binding site for CoA until oxaloacetate binds and makes induced fit to open up CoA binding site after citronylcoa formed in binding site a second induced fit to bring in to split CoA off the citrate these two conformational event use to keep enzyme from simply binding CoA and releasing the acetate group in a useless manner See Figure 16-8

7 7 2. Formation of Isocitrate via cis-aconitate Aconitase or aconitate hydratase 0 ÄG ' = 13.3 kj reversible intermediate usually does not come off the enzyme o At ph 7.4 and 25 C, equilib is 90% citric, 10% isocitrate but in cycle isocitrate is rapidly used up so [] so reaction pulled to right aconitase contains and iron-sulfur cluster (figure 16-10) that acts both in catalysis and for binding Box 16-1 interesting aconitase is also involved in iron regulation

8 8 3. Oxidation of Isocitrate to á-ketoglutarate and CO 2 Isocitrate dehydrogenase Mech figure ÄG ' = kj/mol in eukaryotic cells 2 isozymes of this enzyme Mitochondrial form used NAD + cytosol form uses NADP + cytosol form thought to be used to make NADPH for synthetic pathways Note the CO is NOT a C from the acetate 2 4. Oxidation of á-ketoglutarate to Succinyl-CoA and CO 2 á-ketoglutarate dehydrogenase complex 0 ÄG ' = kj/mol

9 9 virtually identical to pyruvate dehydrogenase complex in both structure and function E 1 enzymes are similar, but not identical, need to binds different substrates E 2 very similar E 3 virtually identical probably share common evolutionary origin 5. Conversion of Succinyl-CoA to succinate S Succinyl-CoA synthase (succinate thiokinase) 0 ÄG ' = -2.6 kj/mol Mechanism figure Pi is used to displace CoA from succinate the phosphoanhydride then phophoylates a His in the active site to generate suc Suc floats off, and the phosphoenzyme then can add phosphate to ADP or GDP enzyme is 2 units á 32,000 binds the CoA and gets phosphorylated at his 246 â 42,000 binds ADP or different â GDP active site at interface substrate level (direct phosphorylation)

10 If make GTP can use nucleoside diphosphate kinase to transfer to ADP for no cost Note: above mechanism is almost exactly the same as one used in pyruvate dehydrogenase and the oxidation of isoleucine, leucine or valine! Figure Oxidation of Succinate to Fumarate 10 Succinate dehydrogenase o ÄG =0 This enzyme tightly bound to inner mitochondrial membrane Only membrane bound enzyme in the whole cycle three different iron sulfur clusters and 1 covalently bound FAD This FAD passes electrons to membrane bound portion of oxidative phosphorylation Will get about 1.5 ATP for this FADH 2 Malonate (not malate) very similar to Succinate (See margin page 647 for structures) acts as competitive inhibitor of this enzyme, and therefor for entire TCA cycle

11 11 7. Hydration of fumarate to malate (Trans) Fumarase (or fumarate hydratase) 0 ÄG ' =-3.8 kj/mole highly stereospecific trans not cis double bond make L not D nd (Will do in lab 2 semester) 8. Oxidation of Malate to Oxaloacetate L-malate dehydrogenase 0 ÄG ' = 29.7 kj/mol very unfavorable under standard state conditions -6 but conc of oxaloacetate in cell extremely low (<10 ) due to initial reaction of TCA cycle so reaction keeps going

12 12 C. Energy in the cycle is efficiently conserved Total for 1 turn of the cycle CH3CO-SCoA in 2 CO 2 Out 3 NADH out 1 FADH 2 out 1 GTP or ATP out The 2 C that appear as CO 2 are NOT the same that came in the acetate Will see in chapter 19 that NADH yields about 2.5 ATP and FADH 2 yields about 1.5 ATP So TCA cycle give 3(2.5) + 1(1.5) + 1 ATP = 10 ATP Lets put the whole total together Chapter 15 1 glucose to 2 pyruvate + 2 ATP + 2 NADH (~2.5 ATP/NADH = 5 ATP) = = 7 ATP 2 Pyruvate to acetyl Co A yield 2 NADH for another 5 ATP And 2 acetyl-coa thru the TCA give us 20 ATP Thus our total is Glucose 6 CO ATP = 32 ATP 2 32 x 30.5 kj/mole = 976 kj of E conserved Glucose to CO 2 could yield 2840 so 976/2840 = 33.3% efficient D. Why is oxidation of acetate so complicated? 8 step cycle to oxidize 2 C seems a bit excessive Nature usually much more economical Job is not simple oxidation but to be hub of metabolism Can use as source or drain of 4, 5, and 6C compounds Also product of evolution, so may not be cleanly designed Some modern anaerobes use incomplete cycle just to synthesize various precursors

13 E. Citric Acid cycle components as intermediates TCA is amphibolic pathway Serves both anabolism and catabolism (build up and tear down) á-ketoglutarate and oxaloacetate simple transamination from glu and asp And other amino acid oxaloacetate can also be converted to glucose in gluconeogenesis Succinyl-CoA intermediate in synthesis of heme ring see figure for summary F. Anaplerotic Reactions if an TCA intermediate removed as a synthesis precursor, then TCA can t carry on because is a cycle Need to replace intermediates when they are siphoned off anapleroic reaction, one that replaces missing intermediates Most common anaplerotic reaction shown in table 16-2 also in red on figure These reactions vary from one tissue to another One of main reactions is carboxylation of pyruvate by pyruvate caboxylase To make oxaloacetate Take an ATP for E Highly regulated Saw this last chapter as way to pyruvate to oxaloacetate to PEP Virtually inactive unless acetylco-a present (Indicating that not enough TCA is around to burn it up) All of the anaplerotic reaction are going to be regulated G. Biotin the mention of pyruvatecarboxylase introduces another vitamins we can look at Biotin Specialized carrier of CO 2 Structure/mech shown in figure In this enzyme covalently attached to end amino group on a lys Making another long flexible arm In enzyme step 1 carboxylation of biotin Step 2 transfer of carboxyl to pyruvate Occur at separate sites to long flex is used to move C from one site to the other Biotin required in human diet, but synthesized by many intestinal bacteria - so deficiency is rare Mostly occurs when eat large amounts of raw eggs 13

14 14 Avidin is an egg protein that binds biotin Once bound can t be absorbed by intestine But if egg is cooked, protein denatured, can t bind 16.3 Regulation of TCA cycle flow from pyruvate to cycle has 2 control points conversion of pyruvate to acetyl Co A Pyruvate dehydrogenase (-33.4 kj) Entry of acetyl CoA into cycle Citrate synthase (-32.3 kj) At first this seems surprising because you have 2 control points in a row but control at other points important because acetyl CoA can have other sources Fatty acid oxidation Some amino acid oxidation and acetyl CoA can go other places FA synthesis for one A. Production of Acetyl CoA by Pyruvate Dehydrogenase both allosteric and covalent modification types of control Complex inhibited by ATP, acetyl-coa, NADH ( reaction products) Inhibition enhance by long-chain fatty acids + activated by AMP, CoA, and NAD which accumulate when too little is introduced to the cycle Net turned off when have lots of E, turned on when E is low In vertebrates additional covalent control Inhibited by reversible phosphorylation of a ser on E 1 by a kinase Kinase turned on by excess ATP P is removed by a specific phophatase Plant may also have phosphorylation control, but not entirely clear E coli definitely not phosphorylation control (on top of allosteric) B. TCA cycle is regulated at its three exergonic steps citrate synthase (-32.2 kj) isocitrate dehydrogenase (-20.9 kj) á-ketoglutarate dehydrogenase (-33.5 kj) see figure #1 by NADH, citrate, ATP ADP #2 by ATP Ca or ADP #3 by succinyl CoA Ca 2+ In general well matched, so nothing accumulates

15 15 C. Substrate Channeling through Multienzyme complexes Originally thought that all enzyme in TCA pathway were simply freely dissolved in the mitochondrial matrix May have simply because that is how we isolated them Growing evidence that many or all are part of multi-enzyme complexes called metabolons So not feely soluble enzymes, but highly organized with channeling of intermediates to make more efficient D. Some mutations in TCA cycle lead to Cancer You can read details if interested 16.4 The Glyoxylate Cycle The two steps PEP to pyruvate and pyruvate to acetyl CoA are strongly exothermic, and essentially irreversible Thus once C has gotten down to the pyruvate or acetyl-coa level, it is committed to oxidation can cannot be recycled back to the gluconeogenesis pathway to make carbohydrates Thus all vertebrates cannot convert fatty acids or acetate into carbohydrates. Plants can do this because they have an additional cycle called the glyoxylate cycle. And this cycle is now discussed. I am going to show that I am a vertebrate chauvinist and I am not going to be politically correct and give the plants their fair time. Thus we will not study this cycle. But if you are a Plant person, this should be required reading.