BIOCHEMISTRY Oxidation of Hexoses by Dr.

Magat 2015B
I. Fates of glucose in different cells 1. Glucose transport 2. Hexokinase: glucose is phosphorylated 3. PPP (Pentose Phosphate Pathway) 4. Glycolysis 5. Lactic acid transport out of the cell 6. Pyruvate decarboxylase 7. TCA Cycle 8. Glycogenesis 9. Glycogenolysis 10. Lipogenesis 11. Formation & release of VLDL 12. Gluconeogenesis 13. Hydrolysis of G6P and release into the blood 14. Glucuronide formation *occurs in different tissues

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Reaction 1 -D-Glucose

-D-Glucose-6

Enzyme: Hexokinase
Irreversible under cellular conditions Exergonic reaction 1 ATP is consumed to prime glucose (C6) Phosphorylation reaction

Reaction 2 -D-Glucose-6 phosphate Phosphate

D-Fructose-6

Enzyme: Phosphohexose/Phosphoglucose

Isomerase Isomerization reaction (Aldehyde to Ketone) Reversible and is not subject to regulation

Reaction 3 D-Fructose-6 Phosphate

Irreversible

D-Fructose-1,6

Enzyme: Phosphofructokinase-1 Uses a 2nd ATP needed to prime glucose (C1) The enzyme of this reaction is the true rate
limiting enzyme of the entire pathway Phosphorylation reaction

**RBC only has the cytoplasm, no mitochondria and ER II. Glycolysis (Embden-Meyerhof Pathway) A. General Features: - Catabolic pathway: 10 metabolic steps - Substrate: D-glucose - requires no 02, but slightly different matter - occurs in the cytoplasm B. Products: 1. 2 ATP 2. NADH electron carrier, proceed to mitochondria for generation of ATP 3. Pyruvate under aerobic conditions (cytoplasm has adequate levels of O2) 4. Lactate under anaerobic conditions C. Types: 1. Aerobic uses O2; pyruvate is the end product 2. Anaerobic uses no O2; lactate is the end product D. Glycolysis can be divided into three (3) stages: Stage 1: Priming Stage 2: Splitting Stage 3: Oxidoreduction-Phosphorylation Stage Priming Splitting OxidoreductionPhosphorylation Product Fructose 1,6bisphosphate Glyceraldehyde 3-Phosphate Pyruvate/Lactate Energy -2 ATP None +4 ATP

Stage 2: Splitting Stage

Reaction 4 D-Fructose-1,6 Bisphosphate phosphate Dihydroxyacetone phosphate

Glyceraldehyde 3+

Enzyme: Aldolase (Fructose-bisphosphate

aldolase) Cleaves 6-carbon molecule into two (2) 3-carbon molecules: G3P & DHAP Reversible reaction Only G3P is needed to proceed in glycolysis so Reaction 5 occurs. Cleavage of C3-C4

Reaction 5 Dihydroxyacetone phosphate phosphate

Glyceraldehyde 3-

Enzyme: Triosephosphate isomerase

Reversible reaction DHAP is converted to G3P Isomerization Reaction Produces 2 molecules of G3P from 1 molecule of Glucose

Stage 3: Oxido-Reduction Phosphorylation Stage

Reaction 6 Glyceraldehyde 3-phosphate

1,3-

Enzyme: Glyceraldehydes 3-phosphate

dehydrogenase

Stage 1: Priming Stage - The priming stage involves input of two molecules of ATP to convert glucose into Fructose 1,6-bisphosphate.

080511 BIOCHEMISTRY 3rd Lecture (2nd LE)

BIOCHEMISTRY Oxidation of Hexoses by Dr. Magat 2015B

Glyceraldehyde 3-phosphate is oxidized to a 1,3
Bisphosphoglycerate with the reduction of NAD+ to NADH. The overall reaction is a coupling reaction of a very favorable exergonic reaction (G3P to 1,3 Bisphosphoglycerate) with an unfavorable endergonic reaction (NAD+ to NADH). Freely Reversible in Cells

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Reaction 7 Phosphoglycerate kinase Enzyme: 1,3-Bisphosphoglycerate 3-phosphoglycerate First site of ATP production in glycolysis

Transfer phosphate from 1,3BPG to ADP (First

substrate level phosphorylation), creating ATP; this ATP is multiplied by 2, since there is two molecules of 1,3-Bisphosphoglycerate. All of the ATP is recovered in this step. Substrate level PO4 reaction A. Pyruvate is reduced to L-lactate by Lactate Dehydrogenase. B. Pyruvate is decarboxylated to Acetyl CoA by Pyruvate Dehydrogenase for the TCA Cycle and other metabolic pathways C. Pyruvate is converted to Oxaloacetate by Pyruvate Carboxylase for Gluconeogenesis D. Pyruvate is converted to Ethanol and Carbon dioxide By Pyruvate Decarboxylase IV. Balance of NAD+ and NADH In anaerobic glycolysis, the generation of NADH and its utilization are coupled (balanced) in the pathway. The two molecules of NADH are generated by glyceraldehyde 3-phosphate dehydrogenase and two are utilized by lactate dehydrogenase. NAD+ is needed for glycolysis to proceed so it is regenerated at the end of the pathway. V. Regulation of Glucose Transport into Cell The rate of entry of glucose into a cell is limited by the number of glucose transporters on the cell surface and the affinity of the transporters for glucose. For most tissues, glucose uptake proceeds at a fairly constant rate, regardless of the amount present in the blood. Liver and pancreatic b cells have a distinct glucose transporter with a high Km. In these cells, then, the amount of incoming glucose is pretty much proportional to the amount of glucose in the blood. This allows the b cells to monitor blood glucose levels directly, and thereby regulate insulin secretion. It also insures that glucose is taken up rapidly by the liver only when it is abundant; in the fasting state, more glucose is delivered to other tissues (particularly the brain). Muscle and fat cells express a third type of glucose transporter. The level of expression is rapidly regulated by insulin. Secretion of insulin causes translocation of these receptors from intracellular stores to the cell surface, allowing more efficient energy storage by these tissues when blood glucose is in excess. VI. Regulation of Glycolysis ENZYME ENHANCED ACTIVTY DECREASED ACTIVITY

Reaction 8 3-phosphoglycerate

2-

Enzyme: Phosphoglycerate mutase

Freely reversible reaction

Isomerization reaction (from Carbon 3 to C2)

Reaction 9 2-phosphoglycerate Phosphoenol

Enzyme: Enolase (dehydrate C2 to produce PEP) Dehydration reaction (Eliminates H2O from 2 phosphoglycerate) This reaction generates a high-energy phosphate from lower energy level.

Reaction 10 Phospoenol pyruvate

Pyruvate

Enzyme: Pyruvate kinase Irreversible under intracellular conditions

Second substrate level phosphorylation ATP is produced; ATP is multiplied by 2 Substrate level PO4 reaction

Reaction 11 (anaerobic) Pyruvate Lactate

Enzyme: Lactate Dehydrogenase

Pyruvate is reduced to L-lactate NADH is oxidized to NAD+ The forward direction of this reaction is the only reaction by which L-lactate can be produced. The reverse direction is the reaction by which Llactate can only be utilized.

* Less energy is produced in anaerobic glycolysis III. Fates of Pyruvate

080511 BIOCHEMISTRY 3rd Lecture (2nd LE)

BIOCHEMISTRY Oxidation of Hexoses by Dr. Magat 2015B

Hexokinase I, II, III PFK I Pyruvate Kinase Glu 6-PO4 AMP, fructose 2,6-BP Fructose 1,6-BP ATP, Citrate, H+ ATP, Alanine

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A. Hexokinase and Glucokinase Step 1 of glycolysis in most tissues, including muscle and brain. Four types of Hexokinase (I, II, III, IV) are expressed in tissue specific manner in the body. Hexokinase I, II, and III are found in most tissues. Liver parenchymal cells and B cells of pancreas are unique in that they contain hexokinase IV aka Glucokinase. Glucokinase is indirectly inhibited by fructose 6phosphate. F6P promotes binding of glucokinase to the regulatory protein thereby inhibiting glucokinase. F6P in effect promotes translocation of GK from the cytosol to the nucleus where GK is inhibited by the inhibitory protein. Increased blood glucose concentration translocates GK back into the cytosol. Glucokinase activity is affected by inhibitory binding protein (GK RP), whose affinity for glucokinase is enhanced by binding fructose-6phosphate and lessened by binding fructose-1phosphate. In the liver, the action of glucokinase is opposed by the action of glucose-6-phosphatase, which hydrolytically removes phosphate in a futile cycle. Glucokinase requires a much higher glucose concentration for maximal activity. Glucokinase is not inhibited by G6P. B. Phosphofructokinase Phosphofructokinase (PFK) catalyzes the ratelimiting step in glycolysis and is the most important control point. First irreversible step, unique to the glycolytic pathway PFK is a major control point for glycolysis. PFK is allosterically inhibited by ATP and citrate, allosterically activated by AMP and F2,6BP. 1. Decreased activity PFK is allosterically inhibited by ATP, so glycolysis is slowed when cellular ATP concentrations are high. Regulated by cellular pH. When cellular lactate is high (usually when oxidative phosphorylation is inhibited), the rate of glycolysis is reduced, preventing further accumulation of intracellular acid. This regulation helps to minimize the risk of lactic cidosis when oxygen is scarce. Glycolysis is also a means of preparing carboncontaining backbones for biosynthetic reactions, in the form of acetyl-CoA. Citrate, an intermediate in the TCA cycle, also inhibits PFK, signaling that biosynthetic precursors are abundant.

2. Enhanced Activity PFK is allosterically activated by high levels of AMP. AMP overcomes the inhibitory effect of ATP. Another allosteric activator of PFK is fructose 2, 6 bisphosphate. F-2,6-BP overcomes the inhibitory effect of ATP. F-2,6-BP is made from F6P by a specific kinase, phosphofructokinase 2 (PFK2) ; controlled by phosphorylation. F-2,6-BP also overcomes the inhibitory effect of ATP. Conversion of F6P to F-2,6-BP is also stimulated by high levels of F6P; example of feedforward stimulation (the opposite of feedback inhibition). Feed forward regulation ensures that intermediates on metabolic pathways do not accumulate uselessly. C. Pyruvate Kinase Pyruvate kinase (3rd regulated enzyme ). Like PFK, pyruvate kinase is regulated both by allosteric effectors and by covalent modification (phosphorylation). Activated by F-1,6-BP in the liver (feedforward stimulation) ATP and alanine (a biosynthetic product of pyruvate) act as allosteric inhibitors of pyruvate kinase. Blood glucose level regulate phosphorylation of pyruvate kinase; High glucagon (low blood sugar) causes phosphorylation, which inactivates enzyme. VII. MOA of Triosphosphate Isomerase (TPI) - converts a ketone to an aldehyde - reversible; near maximal efficiency Dihydroxyacetone phosphate to G3P Transfer the carbonyl carbon from C2 to C1 In the active site of the enzyme Glutamate Histidine STEP 1: then obstruct Glutamates serves as a base which will H+ proton from C1. TPI

STEP 2: Histidine will donate its H+ proton to C2 *Indiole intermediate formation carbon to carbon double bond; important intermediate that needs to be formed in an isomerase enzyme. STEP 3: Glutamate will then act as an acid donating the extra H2 to C2 STEP 4: Histidine will obstruct the H+ from C1 *carbonyl carbon is now formed, and active sites (Glu and His) are regenerated. VIII. MOA of G3P DH (Glyceraldyhyde 3-Phosphate Dehydrogenase) & Inhibitors

G3P DH aim is to phosphorylate C1 Requires NAD+ to generate NADH

In the active site of enzyme:

080511 BIOCHEMISTRY 3rd Lecture (2nd LE)

BIOCHEMISTRY Oxidation of Hexoses by Dr. Magat 2015B

o Cysteine thiol grp o Histidine STEP 1: Cysteine lend its H+ to form a covalent bond with its substrate; to hold substrate in place *hemethioacetal intermediate formation this will undergo redox to form thioesther intermediate STEP 2: H+ is donated to NAD forming NADH, + another H obstructed by Histidine *thioesther intermediate formation **the esther group has only 3 bonding sites occupied after the bond from cysteine is dissolved; this remaining site will easily accept the phosphoryl group from orthophosphate? ***Reaction 6 of Glycolysis formation of 1,3 BPG and generation of NADH Inhibitors inhibit the hemethioacetal intermediate formation 1. Sulfhydryl reagents 2. Iodoacetate 3. Hyperglycemia (PARP) 4. Arsenate IX. MOA: Inhibition of Enolase Fluoride interferes with the way enolase bind with its substrate, 2PG Inhibitors of Glycolysis: application in oncology

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Formation of 5 Carbon Sugars, Some Amino

Acids, Triacylglycerols, Bisphosphoglycerate. and 2,3

A. 2,3 Bisphosphoglycerate * This intermediate does not affect the glycolysis, but on the hemoglobin on the RBC to release O2 molecules to the tissues. B. 2,3 Bisphosphoglycerate Formation from 1,3 BPG STEP 1: the phosphoryl group of 1,3 Bisphosphoglycerate is transferred from C1 to C2 by 2,3 BPG mutase STEP 2: the extra phosphoryl group from C2 is removed by 2,3 BPG phosphatase to return to the glycolytic pathway (3PG) after exhibiting its allosteric effect on RBC

Dihydroxyacetone phosphate to G3P Cancer cells require a lot of energy/nutrients because they are rapidly dividing. o glut produced by cancer cells, then eventually will lead to hypoxia o production of hypoxia inducible factor (HIF) a transcription factor; molecule induces transcription(DNA to RNA) , translation (protein synthesis) o Certain enzymes (HK, G6P DH, TK) will be prone to inhibitors because of structural analogues, hence the pathway is affected. X. Other Functions of Glycolysis

MIP PO4tase is bifunctional *Multiple Inositol Polyphosphate (MIP) phosphatase - converts 2,3 BPG to 2PG (to step 9) C. 2,3 BPG: MOA of Phosphoglycerate Mutase

080511 BIOCHEMISTRY 3rd Lecture (2nd LE)

BIOCHEMISTRY Oxidation of Hexoses by Dr. Magat 2015B

Either lysine(Horton) or histidine(Lehninger)
present in the active site of the enzyme

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Histidine - phosphorylated Lysine form a salt bridge or ionic bond with

carboxyl group of substrate(3PG) to stabilize STEP 1: Histidine lends its phosphoryl group on C2 to form 2,3 BPG **to regenerate active site of enzyme: histidine will take back a phosphoryl group from C3 to form (2PG) and go back to the glycolytic pathway XI. Metabolism of Other Hexoses A. Mannose

Reaction 2 Sorbitol + NAD+ Fructose + NADH Enzyme: Polyol Dehydrogenase

increases osmolarity of lens affects structural organization of crystallins enhances rate of protein aggregation and denaturation not used by other pathways normally, not a problem since aldose reductase is present in small concentration C. Galactose

Reaction 1 Mannose Mannose 6-Phosphate Enzyme: Hexokinase Reaction 2 Mannose 6-Phosphate Fructose 6Phosphate Enzyme: Phosphomannose isomerase Reaction 3 Enters the glycolytic pathway or is converted to glucose-6-phosphate by the gluconeogenic pathway of hepatocytes

Reaction 1 Galactose Galactose 1- Phosphate Enzyme: Galactokinase Reaction 2 Galactose 1-Phosphate UDP-Galactose Enzyme: Galactose 1-phosphate Reaction 3 UDP Galactose UDP Glucose Enzyme: UDP-glucose-4-epimerase Reaction 4 UDP Glucose Glucose 1-Phosphate Glucose 6-Phosphate

Reaction 5 Glucose 1-Phosphate

B. Fructose

Reaction 1 Fructose Fructose 1-Phosphate Enzyme: Fructokinase Reaction 2 Fructose 1-Phosphate Dihydroxyacetone phosphate Enzyme: Aldolase Reaction 3 Dihydroxyacetone Phosphate Glyceraldehyde 3Phosphate Enzyme: Triose Phosphate Isomerase Reaction 4 Enters Glycolysis
B.1 Sorbitol Cataracts

Galactosemia: inability to transform galactose into glucose may be due to cellular deficiency of: a. Galactokinase b. Galactose 1-phosphate uryldyl transferase c. UDP galactose-4-epimerase galactose is reduced to galactitol galactitol initiates cataract formation in the lens and may play a role in CNS damage accumulation of galactose 1-phosphate is responsible for liver failure toxic effects of galactose metabolites disappear when galactose is removed from diet Summary of entry of CHO from diet into glycolysis

Reaction 1 Glucose + NADPH Sorbitol + NADP Enzyme: Aldolase Reductase

080511 BIOCHEMISTRY 3rd Lecture (2nd LE)

BIOCHEMISTRY Oxidation of Hexoses by Dr. Magat 2015B

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IN SUMMARY: 1. Glycolysis products: ATP, NADH, pyruvate/lactate 2. Glucose has different roles in different cells 3. Glycolysis: 10 metabolic steps with key enzymes (inhibitors; regulation) 4. Balance between pyruvate & lactate; NAD+ & NADH 5. Other hexoses (galactose, fructose, & mannose)

undergo oxidation

080511 BIOCHEMISTRY 3rd Lecture (2nd LE)