biochem ch 18 - glycolysis

G3P Dehydrogenase

- enzyme that oxidizes G3P to 1,3-BPG and reduces NAD+ to NADH - 1. function: oxidation-reduction 2. substrate: G3P and NAD+ 3. product: 1,3-BPG and NADH 4. inhibition: iodoacetate (block cysteine SH group on enzyme from reaction)

hydroxylase factor-inhibiting HIF (FIH-1)

- enzyme that regulates HIF through inhibition of HIF's transcriptional activity - active at high oxygen levels - hydroxylates HIF-1α subunit at Asn803

phosphoglycerate kinase

- enzyme that transfers a phosphoryl group from 1,3-bisphosphoglycerate to ADP to form an ATP and 3-Phosphoglycerate (3-PG) 1. function: dephosphorylation 2. substrate: 1,3-BPG and ADP 3. inhibition: high ATP, high 3-PG 4. location: all cells 5. cofactor: mg2+ 6. energy requirement: none 7. activation: none. always active 8. product: 3-PG and ATP (x2 per glucose molecule)

oxidative phosphorylation

- is driven energetically by the transport of electrons from appropriate coenzymes and substrates to oxygen - electrons are transferred from electron donors to electron acceptors such as oxygen, in redox reactions. These redox reactions release energy, which is used to form ATP.

2,3-BPG

- is made by reactions that detour around the phosphoglycerate kinase reaction in step 7 of glycolysis - is an important regulator of hemoglobin - stabilizes the deoxy form of hemoglobin and is primarily responsible for the cooperative nature of oxygen binding by hemoglobin - is formed from 1,3-BPG and 3-PG by bisphosphoglycerate mutase

how does Fructose-2,6-bisphosphate regulate phosphofructokinase ?

- it is a potent allosteric activator that increases the affinity of phosphofructokinase for the substrate fructose-6-phosphate (F6P)

how is the Glycolysis and the citric acid cycle coupled ?

- linked via phosphofructokinase, because citrate, an intermediate in the citric acid cycle, is an allosteric inhibitor of phosphofructokinase. - When the citric acid cycle reaches saturation, glycolysis (which "feeds" the citric acid cycle under aerobic conditions) slows down

Reaction 7 of glycolysis

- phosphoglycerate kinase transfers a phosphoryl group from 1,3-bisphosphoglycerate to ADP to form an ATP and 3-Phosphoglycerate (3-PG) (x2 per glucose molecule) - substrate-level phosphorylation - This reaction "pays off" the ATP debt created by the priming reactions in the first phase - ATPs consumed and produced are equal at this reaction in glycoylsis

enolase

- the enzyme in glycolysis that converts 2-PG to PEP (dehydration) 1. function: dehydration 2. substrate: 2-PG 3. inhibition: fluoride ion in the presence of phosphate 4. location: all cells 5. cofactor: K+ and Mg2+ 6. energy requirement: none 7. activation: none. always active 8. product: PEP and H2O

which 3 enzymes in glycolysis are regulated?

1. hexokinase 2. phosphofructokinase 3. pyruvate kinase

can hexokinase only phosphorylate glucose?

No. hexokinase can phosphorylate a variety of hexose sugars, including glucose, mannose, and fructose

lactic acid fermentation

in the absence of oxygen (anaerobic conditions), pyruvate can be reduced to lactate through oxidation of NADH to NAD+

phosphofructokinase regulation

inhibitors 1. high ATP concentrations (allosteric site) 2. high Citrate concentrations activators 1. high AMP concentrations 2. high Fructose-2,6-bisphosphate concentrations 3. low ATP concentrations (active site)

substrate-level phosphorylation

is a metabolic reaction that results in the formation of ATP or GTP by the direct transfer of a phosphoryl (PO3^2-) group to ADP or GDP from another phosphorylated compound

moonlighting protein

is a single protein that has multiple functions that are not due to gene fusions, multiple RNA splice variants or multiple proteolytic fragments

where does Glucokinase operate in the body and when does it turn on?

location: liver, pancreas when: only turns on when cell is rich in glucose and induced by insulin

Reaction 6 of glycolysis

oxidation of G-3-P to 1,3-BPG and reduction of NAD+ to NADH by G3P Dehydrogenase - Energy yield from converting an aldehyde to a carboxylic acid is used to make 1,3-BPG and NADH - The mechanism involves covalent catalysis and a nicotinamide coenzyme, and it is good example of nicotinamide chemistry

what is hypoxia?

oxygen limitation in body

the glycolysis pathway - 2 phases

pg 571 print this out

what does PEP stand for?

phosphoenolpyruvate

what is the most important regulated enzyme reaction in the glycolysis pathway ?

phosphofructokinase - reaction 3

what technique is used to identify tumors based on the fact that they show high rates of glycolysis

positron emission tomography (PET) - Metabolites labeled with 18F can be taken up by human cells (in the brain, for example) Decay of 18F results in positron emission - Positron-electron collisions produce gamma rays - Detection with gamma ray cameras provides 3D models of tumor extent and location - 2-[18F]fluoro-2-deoxy-glucose, used for this purpose, is a substrate for hexokinase

the step by step process of the first phase of glycolysis (5 reactions)

reactant: 1 glucose 1. Phosphorylation of D-glucose at C-6 with ATP by either hexokinase or glucokinase to produce Glucose-6- phosphate (G6P) 2. Phosphoglucoisomerase Catalyzes the Isomerization of Glucose-6-Phosphate (G6P) to D-Fructose-6-phosphate (F6P) 3. Phosphorylation of F6P at C-1 with ATP by Phosphofructokinase to produce fructose-1,6-bisphosphate (F-1,6-P) 4. cleavage of fructose-1,6-bisphosphate between the C-3 and C-4 carbons by Fructose Bisphosphate Aldolase to yield two triose phosphates - (DHAP) and (G3P) 5. Triose Phosphate Isomerase changes DHAP to G3P products: 2 G3P

How Do Cells Regulate Glycolysis?

regulated through Inhibition of the three key enzymes by allosteric effectors 1. hexokinase 2. phosphofructokinase 3. pyruvate kinase

Isozymes

- each of two or more enzymes with identical function but different structure

Pyruvate Kinase

- enzyme in glycolysis that Converts PEP to pyruvate and makes ATP 1. function: dephosphorylation and enol-keto tautomerization 2. substrate: PEP and ADP 3. inhibition: ATP and acetyl-CoA and alanine 4. location: all cells 5. cofactor: K+ and Mg2+ 6. energy requirement: none 7. activation: AMP, fructose-1,6-bisphosphate (allosteric) 8. product: pyruvate and ATP

prolyl hydroxylases (PHDs)

- enzyme that activates HIF - active at low oxygen levels - hydroxylates HIF-1α subunit at Pro402 and Pro564

Reaction 9 of glycolysis

- Conversion of 2-Phosphoglycerate to PEP (dehydration) by Enolase - The enolase makes a high-energy phosphate in preparation for ATP synthesis in step 10 - "Energy content" of 2-PG and PEP are similar - Enolase just rearranges 2-PG to a form that releases more energy upon hydrolysis

the second phase of glycolysis overview

- Conversion of glyceraldehyde-3-phosphate to pyruvate and coupled formation of 4 ATP and 2 NADH. - 5 steps that convert Metabolic energy of glucose produces 4 ATP - Net ATP yield for glycolysis is two ATP - involves two very high-energy phosphate intermediates 1,3-bisphosphoglycerate (1,3-BPG) Phosphoenolpyruvate (PEP)

Why Are Coupled Reactions Important in Glycolysis?

- Coupled reactions convert some, but not all of the metabolic energy of glucose into ATP - Under cellular conditions, approximately 5% of the energy of glucose is released in glycolysis - Coupled reactions involving ATP hydrolysis are also used to drive the glycolytic pathway - exergonic reactions (favorable) are used to drive endergonic reactions (unfavorble)

hypoxia inducible factor (HIF)

- DNA-binding protein that induces cell response to hypoxia - induce changes in gene expression - is a heterodimer of a constitutive nuclear subunit (HIF-1β) and an inducible α-subunit. Both subunits are basic helix-loop-helix transcription factors that bind to hypoxia-inducible genes, and both subunits exist as a series of isoforms excess O2 - inhibited by hydroxylase factor-inhibiting HIF (FIH-1) limited O2 - activated by prolyl hydroxylases (PHDs)

Warburg Effect

- Otto Warburg observed in 1924 that rapidly proliferating cancer cells metabolize glucose mainly to lactate, even when O2 is plentiful - Lewis Cantley has suggested that this behavior arises because cells need more than ATP - they must synthesize large amounts of nucleotides, amino acids, and lipids This requires lots of NADPH for biosynthesis as well as intermediates for building blocks Cancer cells divert large amounts of glucose to the pentose phosphate pathway to produce NADPH - Signaling proteins and pathways regulate the glycolytic pathway. Cancer cells route up to 90% of acquired glucose and glutamine into lactate and alanine, producing large amounts of NADPH

reaction 2 of glycolysis

- Phosphoglucoisomerase Catalyzes the Isomerization of Glucose-6-Phosphate (G6P) to D-Fructose-6-phosphate (F6P) - requires Mg2+ - Ene-diol intermediate in this reaction isomerization reaction - the intermediate between reactant and product - aldehyde to ketone - aldose to ketose reactant to product - glucose-6-phosphate to fructose-6-phosphate - G6P to F6P

Reaction 8 of glycolysis

- Phosphoglycerate mutase catalyzes a phosphoryl group transfer from 3-PG C-3 to C-2 to make 2-PG why does this occur? It repositions the phosphate to make PEP in the following reaction (enolase) - Note the phospho-histidine intermediates - 2,3-BPG is required to phosphorylate His

phosphofructokinase

- Phosphorylation of F6P - requires ATP and Mg2+ - exergonic reaction - most important regulated reaction in the glycolysis pathway 1. function: phosphorylation 2. substrate: F6P and ATP 3. inhibition: high ATP concentrations, Citrate (allosteric) 4. location: all cells 5. cofactor: mg2+ 6. energy requirement: 1 ATP molecule 7. activation: AMP reverses the inhibition, Fructose-2,6-bisphosphate activation 8. product: fructose-1,6-bisphosphate (F-1,6-P) ATP regulation active site: high ATP affinity allosteric site: low ATP affinity High ATP concentrations = decrease enzyme activity low ATP concentrations = increase enzyme activity

reaction 10 of glycolysis

- Pyruvate Kinase Converts PEP to pyruvate and makes ATP - These two ATP (from one glucose) can be viewed as the "payoff" of glycolysis - Large, negative - ΔG - indicating that this reaction is subject to regulation - Keto-enol equilibrium of pyruvate - The conversion of phosphoenolpyruvate (PEP) to pyruvate may be viewed as involving two steps: phosphoryl transfer, followed by an enol-keto tautomerization. The tautomerization is spontaneous and accounts for much of the free energy change for PEP hydrolysis end of glycolysis

reaction 5 of glycolysis

- Triose Phosphate Isomerase changes DHAP to G3P

what is the enzyme kinetics for phosphofructokinase (PFK) at low ATP?

- activity plot is normal michaelis-menten curve

how is Hexokinase regulated ?

- allosterically inhibited by product glucose-6-P

what is the enzyme kinetics for phosphofructokinase (PFK) at high ATP?

- behaves cooperatively and the activity plot is sigmoid

what are the 2 fates of NADH made from glycolysis?

- can be recycled via aerobic or anaerobic pathways 1. If O2 is available (aerobic conditions), NADH is oxidized in the electron transport pathway, making ATP in oxidative phosphorylation 2. In anaerobic conditions, NADH is oxidized by lactate dehydrogenase (LDH), providing additional NAD+ for more glycolysis

3 results of hypoxia in cells

- can cause changes in gene expression that result in: 1. increased angiogenesis (the growth of new blood vessels) 2. increased synthesis of red blood cells 3. increased levels of some glycolytic enzymes (and thus a higher rate of glycolysis) induced by hypoxia inducible factor (HIF)

phosphoglycerate mutase

- catalyzes a phosphoryl group transfer from 3-PG C-3 to C-2 to make 2-PG 1. function: phosphoryl group transfer 2. substrate: 3-PG 3. inhibition: none 4. location: all cells 5. cofactor: mg2+, 2,3-bisphosphoglycerate 6. energy requirement: none 7. activation: none. always active 8. product: 2-PG - phosphoenzyme intermediates, use 2,3-bisphosphoglycerate as a cofactor, and undergo intermolecular phosphoryl group transfers (in which the phosphate of the product 2-phosphoglycerate is not that from the 3-phosphoglycerate substrate but from 2,3-BPG) - phosphoenzyme, with a phosphoryl group covalently bound to a histidine residue at the active site. This phosphoryl group is transferred to the C-2 position of the substrate to form a transient, enzyme-bound 2,3-bisphosphoglycerate, which then decomposes by a second phosphoryl transfer from the C-3 position of the intermediate to the histidine residue on the enzyme. A

Triose Phosphate Isomerase

- the enzyme that Isomerase DHAP to G3P in step 5 of glycolysis - This reaction makes it possible for both products of the aldolase reaction to continue in glycolysis - The reaction involves an ene-diol mechanism - is a near-perfect enzyme (high catalytic efficency) - a turnover number near the diffusion limit 1. function: Isomerization 2. substrate: DHAP 3. inhibition: none 4. location: all cells 5. cofactor: none 6. energy requirement: none 7. activation: none 8. product: G3P

Fructose bisphosphate aldolase

- the enzyme that cleaves fructose-1,6-bisphosphate between the C-3 and C-4 carbon - facilitate reverse aldol condensation - Animal aldolases are Class I aldolases 1. function: aldolase reaction (cleavage of aldol C-C bond) 2. substrate: fructose-1,6-bisphosphate 3. inhibition: none 4. location: all cells 5. cofactor: none 6. energy requirement: none 7. activation: none 8. product: dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P)

hexokinase

- the generic enzyme that can phosphorylate glucose and other hexoses - allosterically inhibited by product glucose-6-P 1. function: phosphorylation 2. substrate: glucose and other hexoses 3. inhibition: G6P product 4. Km of substrate: 0.03 or 0.3 mM 5. location: all cells 6. cofactor: mg2+ 7. energy requirement: 1 ATP molecule 8. activation: none. always active 9. product: glucose-6-P

fermentation

- the production of ATP energy by reaction pathways in which organic molecules function as donors and acceptors of electrons

Glucokinase

- the specific enzyme that can phosphorylate ONLY D-glucose 1. function: phosphorylation 2. substrate: only D-glucose 3. inhibition: none. usually inactive. 4. Km of substrate: 10mM 5. location: liver and pancreas 6: cofactors: mg2+ 7. energy requirement: 1 ATP molecule 8. activation: induced by insulin 9. product: glucose-6-P

How do cells sense O2?

- through the presence of the molcule HIF and the enzyme that regulates it hydroxylase factor-inhibiting HIF (FIH-1)

first priming reaction

- where a phosphate group is added to glucose using ATP. - This reaction is important for its ability to trap glucose within the cell.

what are the 2 phases of glycolysis?

1. glucose to two glyceraldehyde-3-P's (G3P) 2. glyceraldehyde-3-P's to pyruvate's

reaction 4 of glycolysis

-cleavage of fructose-1,6-bisphosphate between the C-3 and C-4 carbons by Fructose Bisphosphate Aldolase to yield two triose phosphates. - The products are dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P)

the net reaction of glycolysis

1 glucose + 2ADP + 2[NAD+] + 2Pi -> 2 pyruvate + 2 ATP + 2 NADH + 2[H+] + 2H2O

what are the 3 fates of Pyruvate From Glycolysis

1. (aerobic conditions) pyruvate is converted to acetyl-coenzyme A, which is then oxidized in the TCA cycle to produce CO2. 2. (anaerobic conditions) pyruvate can be converted to lactate. 3. Alcoholic fermentation in yeast converts pyruvate to ethanol and CO2

Why is glycolysis so important to organisms?

1. For some tissues (such as brain, kidney medulla, and rapidly contracting skeletal muscles) and for some cells (such as erythrocytes and sperm cells), glucose is the only source of metabolic energy. 2. the product of glycolysis— pyruvate—is a versatile metabolite that can be used in several ways

what is the order of the enzymes in the first 5 steps of glycolysis?

1. Hexokinase / glucokinase 2. Phosphoglucoisomerase 3. phosphofructokinase-1 4. fructose bisphosphate aldolase 5. triose phosphate isomerase (TPI)

how does Fructose-2,6-bisphosphate increase the net flow of glucose through glycolysis?

1. allosteric activator for phosphofructokinase 2. inhibitor for fructose-1,6-bisphosphatase

what are the Advantages of Phosphorylating Glucose in reaction one?

1. keeps the substrate in the cell Glucose = neutral molecule so could diffuse across the cell membrane Glucose-6-phosphate = negatively charged = trapped inside cell (membrane is impermeable to charged molecules) 2. rapid conversion of glucose to glucose-6-phosphate keeps the intracellular concentration of glucose low, favoring diffusion of glucose into the cell 3. regulatory control can be imposed only on reactions not at equilibrium, the favorable thermodynamics of this first reaction makes it an important site for regulation

Why does reaction 2 - isomerization of G6P occur in glyclyosis?

1. next step 3 (phosphorylation at C-1) would be tough for hemiacetal -OH, but easy for primary -OH 2. the isomerization to fructose (with a carbonyl group at position 2 in the linear form) activates C-3, facilitating C-C bond cleavage in the fourth step of glycolysis

reaction 1 of glycolysis

1. phosphorylation of D-glucose at C-6 by either hexokinase or glucokinase - REQUIRES 1 ATP - REQUIRES Magnesium ion (Mg2+) - This is a priming reaction - ATP is consumed here in order to get more later - ATP makes the phosphorylation of glucose spontaneous - regulated by allosteric inhibition of hexokinase by G6P

what are the products of glycolysis?

1. pyruvate 2. ATP 3. NADH

what are the 3 products of glycolysis?

1. pyruvate 2. ATP 3. NADH

how many reaction steps in glycolysis?

10

how many G3P does 1 glucose molecule produce?

2

what is the products of the first phase of glycolysis?

2 G3P molecules

How Do Cells Respond to Hypoxic Stress?

Glycolysis - anaerobic pathway (no O2 required) tricarboxylic acid cycle (TCA) - aerobic pathway (O2 required) 1. excess O2 environment - cells prefer to combine these pathways in aerobic metabolism 2. limited O2 environment - cells adapt to carry out more glycolysis Hypoxia - causes changes in gene expression that increases levels of glycolytic enzymes - A trigger for this is a DNA-binding protein called hypoxia inducible factor (HIF) HIF is regulated at high oxygen levels by hydroxylase factor-inhibiting HIF (FIH-1)

what are the 2 distinct but important functions of Phosphoglucoisomerase?

INSIDE the CELL - primary function 1. Isomerization of Glucose-6-Phosphate (G6P) to D-Fructose-6-phosphate (F6P) in reaction 2 of glycolysis OUTSIDE the CELL - moonlighting 2. nerve growth factor, autocrine motility factor (AMF), and differentiation and maturation mediator (DMM)

reaction 3 of glycolysis

Phosphorylation of F6P at C-1 with ATP by Phosphofructokinase to produce fructose-1,6-bisphosphate (F-1,6-P)

first phase of glycolysis - overview

Phosphorylation of glucose and conversion to 2 molecules of glyceraldehyde-3-phosphate; 2 ATPs are used to prime these reactions - glucose will be phosphorylated at C-1 and C-6, and the six-carbon skeleton of glucose will be cleaved to yield two three-carbon molecules of glyceraldehyde-3-phosphate. purpose: Phosphorylation and cleavage reorganize the glucose molecule so that molecules of ATP can be produced in the second phase of glycolysis.

All are important reasons to phosphorylate glucose in the first step of glycolysis EXCEPT: a. the large positive free energy is important in getting the pathway started. b. glucose-6-phosphate has a negative charge preventing transport out of the cell. c. the concentration of free glucose in the cell is lowered favoring influx of glucose. d. phosphorylation keeps the glucose in the cell. e. regulatory control can be imposed only at a reaction not at equilibrium.

a. the large positive free energy is important in getting the pathway started.

why is hexokinase, phosphofructokinase, and pyruvate kinase reactions the sites of regualation in glycolysis?

all 3 enzymes exhibit large negative ΔG values under cellular conditions

is glycolysis an aerobic or anaerobic pathway?

anaerobic - does not require O2

For the first five steps of glycolysis, the appropriate sequence of enzymes is: A. phosphofructokinase-1 (PFK-1). B. hexokinase / glucokinase. C. fructose bisphosphate aldolase. D. Phosphoglucoisomerase. E. triose phosphate isomerase (TPI). a. A, C, B, E, D b. B, C, D, E, A c. B, D, C, A, E d. B, D, A, C, E e. B, D, E, C, A

d. B, D, A, C, E

bisphosphoglycerate phosphatase

enzyme that turns 2,3-BPG to 3-PG

adenylate kinase

enzyme which catalyzes the reaction ADP + ADP <-> ATP + AMP - help control AMP levels and regulate phosphofructokinase

mutase

enzymes that catalyze migration of a functional group within a substrate molecule

tumors show high rates of ____________

glycolysis

hexokinase vs. Glucokinase

hexokinase 1. function: phosphorylation 2. substrate: glucose and other hexoses and ATP 3. inhibition: G6P product 4. Km of substrate: 0.03 or 0.3 mM 5. location: all cells 6. cofactor: mg2+ 7. energy requirement: 1 ATP molecule 8. activation: none. always active 9. product: glucose-6-P Glucokinase 1. function: phosphorylation 2. substrate: only D-glucose and ATP 3. inhibition: none. usually inactive. 4. Km of substrate: 10mM 5. location: liver and pancreas 6: cofactors: mg2+ 7. energy requirement: 1 ATP molecule 8. activation: induced by insulin 9. product: glucose-6-P

what molecule links glycolytic activity to oxygen level?

hypoxia inducible factor (HIF)

what molecule triggers the cellular response to hypoxia?

hypoxia inducible factor (HIF)

Phosphoglucoisomerase

the enzyme that Catalyzes the Isomerization of Glucose-6-Phosphate (G6P) to D-Fructose-6-phosphate (F6P) - requires Mg2+ 1. function: Isomerization 2. substrate: Glucose-6-Phosphate (G6P) 3. inhibition: none 4. location: all cells 5. cofactor: mg2+ 6. energy requirement: none 7. activation: none. always active 8. product: D-Fructose-6-phosphate (F6P) - also a moonlighting protein - Secreted by white blood cells, involved in growth and motion of many types of cells.

bisphosphoglycerate mutase

turns 1,3-BPG into 2,3-BPG * The enzyme that forms 2,3-BPG from 1,3-BPG requires 3-phosphoglycerate. The reaction is actually an intermolecular phosphoryl transfer from C-1 of 1,3-BPG to C-2 of 3-PG to make 2,3-BPG

what are the 2 fates of pyruvate made from glycolysis?

two possible fates: Aerobic or anaerobic pathways 1. In aerobic conditions, pyruvate proceeds through the tricarboxylic acid (TCA) cycle 2. Anaerobic metabolism of pyruvate leads to lactate (in microorganisms and animals) or ethanol (in yeast)

how does Hexokinase Binds Glucose and ATP ?

with an Induced Fit

Are Substrates Other Than Glucose Used in Glycolysis?

yes. - Sugars other than glucose can be glycolytic substrates ex. Fructose, mannose and galactose can all be used glycerol

glycolysis overview

—the stepwise degradation of glucose - is also called the Embden-Meyerhof (or Warburg) Pathway - All cells carry out this reaction - Ten reactions steps Two phases: 1. glucose to two glyceraldehyde-3-P's 2. glyceraldehyde-3-P's to pyruvate's - Products are pyruvate, ATP and NADH - Three possible fates for pyruvate