Krebs Cycle. Dr. Leena S Barhate

Transcription

1 Krebs Cycle Dr. Leena S Barhate

2 Acknowledgement www2.fiu.edu/~bch3033/handouts/lh6ch16t CA.ppt ppt cronus.uwindsor.ca/units/biochem/web/bioch emi.nsf/.../citric%20acid%20cycle.ppt

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4 Discovered CAC in Pigeon Flight Muscle

5 The Citric acid cycle It is called the Krebs cycle or the tricarboxylic and is the hub of the metabolic system. It accounts for the majority of carbohydrate, fatty acid and amino acid oxidation. It also accounts for a majority of the generation of these compounds and others as well. Amphibolic - acts both catabolically and anabolically 3NAD+ + FAD + GDP + Pi + acetyl-coa 3NADH + FADH + GTP + CoA + 2C 2

6 1937: Krebs: Enzymatic conversion of Pyruvate + xaloacetate to citrate and C 2 Discovered the cycle of these reactions and found it to be a major pathway for pyruvate oxidation in muscle.

7 In Cytosol In Mitochondria

8 Reaction of pyruvate dehydrogenase complex (PDC) Thiamine pyrophosphate Flavin adenine dinucleotide Pyruvate dehydrogenase Dihydrolipoyl transacetylase Dihydrolipoyl dehydrogenase

9 Acetyl-lipoamide

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11 Pyruvate dehydrogenase Complex (PDC) It is a multi-enzyme complex containing three enzymes associated together noncovalently: E-1 : Pyruvate dehydrogenase, uses Thiamine pyrophosphate as cofactor bound to E1 E-2 : Dihydrolipoyl transacetylase, Lipoic acid bound, CoA as substrate E-3 : Dihydrolipoyl Dehydrogenase FAD bound, NAD + as substrate Advantages of multienzyme complex: 1. Higher rate of reaction: Because product of one enzyme acts as a substrate of other, and is available for the active site of next enzyme without much diffusion. 2. Minimum side reaction. 3. Coordinated control.

12 The Krebs Cycle ccurs in the matrix of the mitochondrion Aerobic phase (requires oxygen) 2-carbon acetyl CoA joins with a 4-carbon compound to form a 6- carbon compound called Citric acid

13 Citric acid (6C) is gradually converted back to the 4- carbon compound -ready to start the cycle once more The carbons removed are released as C 2 -enzymes controlling this process called decarboxylases The hydrogens, which are removed, join with NAD to form NADH2 -enzymes controlling the release of hydrogen are called dehydrogenases

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18 Citric Acid Cycle

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20 Summary of Krebs Cycle How many energy-producing molecules do we have per 1 glucose molecule? Intermediate step: Pyruvate oxidation 1 NADH x2= 2 NADH Krebs Cycle: 3 NADHx2= 6 NADH 1 ATPx2= 2 ATP 1 FADH2x2= 2 FADH2 + 2 ATP, 2 NADH from glycolysis Total: 4 ATP, 10 NADH, 2 FADH2 --> forms 38 ATP in the electron transport chain

21 ATP

22 ATP

23 ATP

24 ATP

25 Gluconeogenesis;

26 glucose glycolysis gluconeogenesis pyruvate Lactate Amino acid glycerol gluco neo genesis sugar (re)new make/ create

27 The source of pyruvate and oxaloacetate for gluconeogenesis during fasting or carbohydrate starvation is mainly amino acid catabolism. Some amino acids are catabolized to pyruvate, oxaloacetate, or precursors of these. Muscle proteins may break down to supply amino acids. These are transported to liver where they are deaminated and converted to gluconeogenesis inputs. Glycerol, derived from hydrolysis of triacylglycerols in fat cells, is also a significant input to gluconeogenesis. 27

28 Noncarbohydrate precursors of glucose Fatty acids Triglycerols glycerol Dietary & muscle proteins Amino acids 28

29 Main sites of gluconeogenesis: Major site: Liver. Minor site: Kidney. Very little: Brain. Muscle (skeletal and heart). In liver and kidney it helps to maintain the glucose level in the blood so that brain and muscle can extract sufficient glucose from it to meet their metabolic demands. 29

30 Gluconeogenesis Versus Glycolysis: 7 steps are shared between glycolysis and gluconeogenesis. 3 essentially irreversible steps shift the equilibrium far on the side of glycolysis. Most of the decrease in free energy (consuming energy) in glycolysis takes place during these 3 steps. These steps must be bypassed in Gluconeogenesis. Two of the bypass reactions involve simple hydrolysis reactions. 30

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32 6 C H 2 P 3 2 G lu c o s e -6 -p h o s p h a ta s e C H 2 H H 4 5 H H H H 1 H 2 H H H H H + P i H 3 2 H H H H H g lu c o s e -6 -p h o s p h a te H g lu c o s e H Hexokinase or Glucokinase (Glycolysis) catalyzes: glucose + ATP glucose-6-phosphate + ADP Glucose-6-Phosphatase (Gluconeogenesis) catalyzes: glucose-6-phosphate + H 2 glucose + P i

33 6 C H 2 P 3 2 G lu c o s e -6 -p h o s p h a ta s e C H 2 H H 4 5 H H H H 1 H 2 H H H H H + P i H 3 2 H H H H H g lu c o s e -6 -p h o s p h a te H g lu c o s e H Glucose-6-phosphatase enzyme is embedded in the endoplasmic reticulum (ER) membrane in liver cells. The catalytic site is found to be exposed to the ER lumen. Another subunit may function as a translocase, providing access of substrate to the active site.

34 P h o s p h o fru c to k in a s e 6 2 C H 2 P 3 1C H 2 H 6 2 C H 2 P 3 A T P A D P 2 1 C H 2 P 3 5 H H 2 5 H H 2 H 4 3 H H H P i H 4 3 H H 2 H H fru c to se -6 -p h o s p h a te fru c to se -1,6 -b is p h o s p h a te F ru c to s e -1,6 -b io s p h o s p h a ta s e Phosphofructokinase (Glycolysis) catalyzes: fructose-6-p + ATP fructose-1,6-bisp + ADP Fructose-1,6-bisphosphatase (Gluconeogenesis) catalyzes: fructose-1,6-bisp + H 2 fructose-6-p + P i

35 Bypass of Pyruvate Kinase: Pyruvate Kinase (last step of Glycolysis) catalyzes: phosphoenolpyruvate + ADP pyruvate + ATP For bypass of the Pyruvate Kinase reaction, cleavage of 2 ~P bonds is required. DG for cleavage of one ~P bond of ATP is insufficient to drive synthesis of phosphoenolpyruvate (PEP). PEP has a higher negative DG of phosphate hydrolysis than ATP.

36 P y ru v a te C a rb o x y la s e P E P C a rb o x y k in a s e C C A T P A D P + P i C G T P G D P C C HC CH 3 3 CH 2 C 2 C P 3 C 2 CH 2 p y ru v a te o x a lo a c e ta te P E P Bypass of Pyruvate Kinase (2 enzymes): Pyruvate Carboxylase (Gluconeogenesis) catalyzes: pyruvate + HC 3 + ATP oxaloacetate + ADP + P i PEP Carboxykinase (Gluconeogenesis) catalyzes: oxaloacetate + GTP PEP + GDP + C 2

37 P y ru v a te C a rb o x y la s e P E P C a rb o x y k in a s e C C A T P A D P + P i C G T P G D P C C CH 2 C P 3 2 CH 3 HC 3 C C 2 CH 2 p y ru v a te o x a lo a c e ta te P E P Contributing to spontaneity of the 2-step process: Free energy of one ~P bond of ATP is conserved in the carboxylation reaction. Spontaneous decarboxylation contributes to spontaneity of the 2nd reaction. Cleavage of a second ~P bond of GTP also contributes to driving synthesis of PEP.

38 Pyruvate Carboxylase uses biotin as prosthetic group. lysine H 3 N + C C H C H 2 C H 2 C H 2 C H 2 N H 3 N s u b je c t to c a rb o x y la tio n H N H 2 C C H C S b io tin C H N H C H (C H 2 ) 4 C N H (C H 2 ) 4 C H Biotin has a 5-C side chain whose terminal carboxyl is in amide linkage to the e-amino group of an enzyme lysine. The biotin & lysine side chains form a long swinging arm that allows the biotin ring to swing back & forth between 2 active sites. ly s in e re s id u e C N H

39 P C H carboxyphosphate - C C N N H C H C H H 2 C C H C S (C H 2 ) 4 C N H (C H 2 ) 4 C H c a rb o x yb io tin ly s in e re s id u e N H Biotin carboxylation is catalyzed at one active site of Pyruvate Carboxylase. ATP reacts with HC 3 to yield carboxyphosphate. The carboxyl is transferred from this ~P intermediate to N of a ureido group of the biotin ring. verall: biotin + ATP + HC 3 carboxybiotin + ADP + P i

40 At the other active site of Pyruvate Carboxylase the activated C 2 is transferred from biotin to pyruvate: C C C H 3 p y ru v a te - C C N N H c a rb o x y b io tin C H C H H 2 C C H S ( C H 2 ) 4 C N H R carboxybiotin + pyruvate biotin + oxaloacetate C C C H 2 C C H N N H C H C H H 2 C C H b io tin View an animation. o x a lo a c e ta te S ( C H 2 ) 4 C N H R

41 Pyruvate Carboxylase (pyruvate oxaloactate) is allosterically activated by acetyl CoA. [xaloacetate] tends to be limiting for Krebs cycle. G lu c o n e o g e n e s is o x a lo a c e ta te G lucose-6 -p h o s p h a ta s e glucose-6 -P pyruvate a c e ty l C o A K rebs C ycle G lycolysis citrate g lu c o s e fatty acids ketone bodies When gluconeogenesis is active in liver, oxaloacetate is diverted to form glucose. xaloacetate depletion hinders acetyl CoA entry into Krebs Cycle. The increase in [acetyl CoA] activates Pyruvate Carboxylase to make oxaloacetate.

42 P E P C a rb o x y k in a s e R e a c tio n C C C G T P G D P C C H 2 C C P 3 2 C C 2 C H 2 C H 2 o x a lo a c e ta te P E P PEP Carboxykinase catalyzes GTP-dependent oxaloacetate PEP. It is thought to proceed in 2 steps: xaloacetate is first decarboxylated to yield a pyruvate enolate anion intermediate. Phosphate transfer from GTP then yields phosphoenolpyruvate (PEP).

43 In the bacterial enzyme, ATP is P i donor instead of GTP. In this crystal structure of an E. Coli PEP Carboxykinase, pyruvate is at the active site as an analog of PEP/ oxaloacetate. ATP Mg++ PEP Carboxykinase active site ligands Mn ++ pyruvate PDB 1AQ2 A metal ion such as Mn ++ is required for the PEP Carboxykinase reaction, in addition to a Mg ++ ion that binds with the nucleotide substrate at the active site. Mn ++ is thought to promote P i transfer by interacting simultaneously with the enolate oxygen atom and an oxygen atom of the terminal phosphate of GTP or ATP.

44 g ly c e ra ld e h y d e -3 -p h o s p h a te NAD + + P i NADH + H + G ly c e ra ld e h y d e -3 -p h o s p h a te D e h y d ro g e n a s e 1,3 -b is p h o s p h o g ly c e ra te Summary of Gluconeogenesis Pathway: Gluconeogenesis enzyme names in red. Glycolysis enzyme names in blue. ADP P h o s p h o g ly c e ra te K in a s e A T P 3 -p h o s p h o g ly c e ra te P h o s p h o g ly c e ra te M u ta s e 2 -p h o s p h o g ly c e ra te H 2 E n o la s e p h o s p h o e n o lp y ru v a te C 2 + G D P P E P C a rb o x y k in a s e G T P o x a lo a c e ta te P i + A D P H C 3 + A T P p y ru v a te P y ru v a te C a rb o x y la s e G lu c o n e o g e n e s is

45 glucose G lu c o n e o g e n e s is P i H 2 G lu c o s e -6 -p h o s p h a ta s e g lu c o s e -6 -p h o s p h a te P h o s p h o g lu c o s e Is o m e ra s e fru c to s e -6 -p h o s p h a te P i H 2 fru c to s e -1,6 -b is p h o s p h a te A ld o la s e F ru c to s e -1,6 -b is p h o s p h a ta s e g ly c e ra ld e h y d e -3 -p h o s p h a te + d ih y d ro x y a c e to n e -p h o s p h a te (continued) T rio s e p h o s p h a te Isom erase

46 Glycolysis & Gluconeogenesis are both spontaneous. If both pathways were simultaneously active in a cell, it would constitute a "futile cycle" that would waste energy. Glycolysis: glucose + 2 NAD ADP + 2 P i 2 pyruvate + 2 NADH + 2 ATP Gluconeogenesis: 2 pyruvate + 2 NADH + 4 ATP + 2 GTP glucose + 2 NAD ADP + 2 GDP + 6 P i Questions: 1. Glycolysis yields how many ~P? 2 2. Gluconeogenesis expends how many ~P? 6 3. A futile cycle of both pathways would waste how many ~P per cycle? 4

47 P h o s p h o fru c to k in a s e 6 2 C H 2 P 3 1C H 2 H 6 2 C H 2 P 3 A T P A D P 2 1 C H 2 P 3 5 H H 2 5 H H 2 H 4 3 H H H fru c to se -6 -p h o s p h a te P i H 2 fru c to se -1,6 -b is p h o s p h a te To prevent the waste of a futile cycle, Glycolysis & Gluconeogenesis are reciprocally regulated. Local Control includes reciprocal allosteric regulation by adenine nucleotides. Phosphofructokinase (Glycolysis) is inhibited by ATP and stimulated by AMP. Fructose-1,6-bisphosphatase (Gluconeogenesis) is inhibited by AMP. H 4 3 H F ru c to s e -1,6 -b io s p h o s p h a ta s e H H

48 The opposite effects of adenine nucleotides on Phosphofructokinase (Glycolysis) Fructose-1,6-bisphosphatase (Gluconeogenesis) insures that when cellular ATP is high (AMP would then be low), glucose is not degraded to make ATP. When ATP is high it is more useful to the cell to store glucose as glycogen. When ATP is low (AMP would then be high), the cell does not expend energy in synthesizing glucose.

49 Global Control in liver cells includes reciprocal effects of a cyclic AMP cascade, triggered by the hormone glucagon when blood glucose is low. Phosphorylation of enzymes & regulatory proteins in liver by Protein Kinase A (camp Dependent Protein Kinase) results in inhibition of glycolysis stimulation of gluconeogenesis, making glucose available for release to the blood.

50 Enzymes relevant to these pathways that are phosphorylated by Protein Kinase A include: Pyruvate Kinase, a glycolysis enzyme that is inhibited when phosphorylated. CREB (camp response element binding protein) which activates, through other factors, transcription of the gene for PEP Carboxykinase, leading to increased gluconeogenesis. A bi-functional enzyme that makes and degrades an allosteric regulator, fructose-2,6-bisphosphate.

51 P F K A c ti v i ty Recall that Phosphofructokinase, the rate-limiting step of Glycolysis, is allosterically inhibited by ATP. At high concentration, ATP binds at a low-affinity regulatory site, promoting the tense conformation. 60 Sigmoidal dependence of reaction rate on [fructose-6- phosphate] is observed at high [ATP] lo w [A T P ] h ig h [A T P ] [F r u c to s e -6 -p h o s p h a te ] m M

52 P F K A c ti v i ty 60 PFK activity in the presence of the globally controlled allosteric regulator fructose-2,6- bisphosphate is similar to that at low ATP lo w [A T P ] h ig h [A T P ] [F r u c to s e -6 -p h o s p h a te ] m M Fructose-2,6-bisphosphate promotes the relaxed state, activating Phosphofructokinase even at high [ATP]. Thus activation by fructose-2,6-bisphosphate, whose concentration fluctuates in response to external hormonal signals, supersedes local control by [ATP].

53 P F K 2 /F B P a s e 2 h o m o d im e r P D B 2 B IF The allosteric regulator fructose-2,6-bisphosphate is synthesized & degraded by a bi-functional enzyme that includes 2 catalytic domains: PFK-2 d o m a in F B P a s e -2 d o m a in w ith b o u n d fru c to s e -6 -P Phosphofructokinase-2 (PFK2) domain catalyzes: Fructose-6-phosphate + ATP fructose-2,6-bisphosphate + ADP Fructose-Biophosphatase-2 (FBPase2) domain catalyzes: Fructose-2,6-bisphosphate + H 2 fructose-6-phosphate + P i Bifunctional PFK2/FBPase2 assembles into a homodimer.

54 P F K 2 /F B P a s e 2 h o m o d im e r P D B 2 B IF PFK-2 d o m a in F B P a s e -2 d o m a in w ith b o u n d fru c to s e -6 -P Adjacent to the PFK-2 domain in each copy of the liver enzyme is a regulatory domain subject to phosphorylation by camp-dependent Protein Kinase. Which catalytic domains of the enzyme are active depends on whether the regulatory domains are phosphorylated.

55 (a c tiv e a s P h o s p h o fru c to k in a s e -2) E n z - H A T P A D P fru c to s e -6 -P fru c to s e -2,6 -b is P P i E n z - -P 3 2 (a c tiv e a s F ru c to s e -B is p h o s p h a ta s e -2) View an animation. camp-dependent phosphorylation of the bi-functional enzyme activates FBPase2 and inhibits PFK2. [Fructose-2,6-bisphosphate] thus decreases in liver cells in response to a camp signal cascade, activated by glucagon when blood glucose is low.

56 (a c tiv e a s P h o s p h o fru c to k in a s e -2) E n z - H A T P A D P Downstrea m effects of the camp cascade: fru c to s e -6 -P fru c to s e -2,6 -b is P P i 2 E n z - -P 3 (a c tiv e a s F ru c to s e -B is p h o s p h a ta s e -2) Glycolysis slows because fructose-2,6-bisphosphate is not available to activate Phosphofructokinase. Gluconeogenesis increases because of the decreased concentration of fructose-2,6-bisphosphate, which would otherwise inhibit the gluconeogenesis enzyme Fructose- 1,6-bisphosphatase.

57 G ly c o g e n P y ru v a te X G lu c o n e o g e n e s is G lu c o s e -1 -P G lu c o s e -6 -P G lu c o s e + P i X G lu c o s e -6 -P a s e G ly c o ly s is P a th w a y Summary of effects of glucagon-camp cascade in liver: Gluconeogenesis is stimulated. Glycolysis is inhibited. Glycogen breakdown is stimulated. Glycogen synthesis is inhibited. Free glucose is formed for release to the blood.

58 Energetics of Gluconeogenesis figure 13-1 Pyruvate Carboxylase 2 ATPs PEP Carboxykinase 2 GTPs 3-P-glycerate kinase 2 ATPs Glyceraldehyde-3-P dehydrogenase 2NADH

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61 Glyoxylate cycle Hans Kornberg and Neil Madsen The glyoxylate cycle results in the net conversion of two acetyl-coa to succinate instead of 4 C 2 in citric acid cycle. Succinate is transferred to mitochondrion where it can be converted to AA (TCA) Can go to cytosol where it is converted to oxaloacetate for gluconeogenesis. Net reaction 2Ac-CoA + 2NAD + + FAD AA + 2CoA + 2NADH +FADH 2 + 2H + Plants are able to convert fatty acids to glucose through this pathway

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66 Glyoxylate cycle The glyoxylate cycle results in the net conversion of two acetyl-coa to succinate instead of 4 C 2 in citric acid cycle. Succinate is transferred to mitochondrion where it can be converted to AA (TCA) Can go to cytosol where it is converted to oxaloacetate for gluconeogenesis. Net reaction 2Ac-CoA + 2NAD + + FAD AA + 2CoA + 2NADH +FADH 2 + 2H + Plants are able to convert fatty acids to glucose through this pathway

67 Reference cronus.uwindsor.ca/units/biochem/web/.../citric%20acid% 20cycle.ppt dcycle.ppt 0Cycle.ppt www2.fiu.edu/~bch3033/handouts/lh6ch16tca.ppt