Biochemistry Exam 3

lectures 21-22

Gluco

glucose

neo

new

genesis

generation

gluconeogensis

the generation of glucose from non carbohydrate resources

what do some tissues exclusively rely on for energy generation?

glycolysis

where does gluconeogenesis occur?

mainly in the liver, less in the kidneys

what can deplete glycogen stores?

starvation, exercise, and long fasts

glycogen

storage form of glucose

glucogenic molecules

can be converted into pyruvate or oxaloacetate

important precursurs for gluconeogenesis

lactate, pyruvate, triacylglycerol, and amino acids like alanine, aspartate, glutamate

Bypass reactions

irreversible reactions in gluconeogenesis

how many steps of gluconeogenesis ARE the reverse of glycolysis, and why are the rest not?

7 are reversible, the other 3 have very negative delta G values

gluconeogenesis is not

the reverse of glycolysis

what are the irreversible reactions in glycolysis

• glucose to glucose-6-phosphate by hexokinase

• fructose-6-phosphate to fructose 1-6 bisphosphate by phosphofuctokinase-1

• phosphoenol pyruvate to pyruvate by pyruvate kinase

both glycolysis or gluconeogenesis are…and what are the implications?

irreversible, when a cell is doing one process it cannot be doing the other

How do the delta G differ in gluconeogenesis conditions

they get even more negative for ATP using steps

The last step of glycolysis

the conversion of phosphoenol pyruvate to pyruvate coupled with the generation of ATP, using pyruvate kinase; very favorable because phosphoenolpyruvate is very unstable

what does a sugar requre

3 carbons, a hydroxyl group, and a carbonyl group

Oxaloacetate

4 carbon, highly acidic molecule

the first bypass reaction

• pyruvate to phosphopenolpyruvate

• pyruvate is transported to the mitochondria

Pyruvate to oxaloacetate

• the enzyme pyruvate carboxylase uses the hydrogen atom from a bicarbonate and an ATP molecule to incorporate CO2 into the pyruvate

what are we also transporting during the malate shuttle?

NAD+

how does oxaloacetate get transported into the cytosol?

• it has no transporters so it gets converted into L-malate through the use of malate dehydrogenase that reduces the oxaloacetate and NADH becomes NAD+

• in the cytosol, the malate turns back into oxaloacetate by reversing the process and malate dehydrogenase

• MDH reaction

where do you find oxaloacetate?

in the mitochondria

How exactly does pyruvate carboxylase work?

• at site 1, bicarbonate is converted to CO2 using ATP, then biotin binds and picks up the CO2

• Biotin binds to the lysine arm using an amide linkage

• using the lysine arm, the CO2 is moved to pyruvate in site 2 by biotin, forming oxaloacetate

Pyruvate carboxylase structure

• two active sites with a lysine residue between them

Conversion of OAA to PEP in the cytosl

• OAA uses a GTP to make PEP, CO2, and GDP

• Uses PEP carboxykinase for both the decarboxylation and the phosphorylation at the carbon 2 position

Expenditure to make one molecule of phosphoenolpyruvate

phase 1 uses 1 ATP, phase 2 uses one GTP

Why is Pep being made during gluconeogenesis even though delta G is positive?

in gluconeogenic conditions, pep is readily consumed by glycolysis in the cytosol and the concentration gets lower, pushing the reaction in the forward direction with a delta G of -25

importance of using the malate shuttle?

• NADH in the cytosol is 7 times lower than in the mitochondria

• malate shuttle also moves NADH from the mitochondria to the cytosol for subsequent gluconeogenesis reactions

What happens if we use lactate as a starting point for gluconeogenesis instead of pyruvate?

• Lactate is converted into pyruvate in the cytosol by lactate dehydrogenase (LDH), producing the cytosolic NADH needed for gluconeogenesis.

• Pyruvate enters the mitochondria and is converted into oxaloacetate by pyruvate carboxylase.

• Oxaloacetate is converted directly into PEP by mitochondrial PEP carboxykinase, and PEP exits into the cytosol

Why is the malate shuttle unnecessary for lactose?

lactate oxidation already provides cytosolic NADH, so oxaloacetate does not need to be converted into malate to transfer reducing equivalents

Mitochondrial vs cytosolic PEP carboxykinase

encoded by different genes but encode the same reaction

which reactions are reversible?

from phosphoenolpyruvate to fructose 1-6 bisphosphate

the second bypass reaction

dephosphorylation of Fructose 1-6 bisphosphate

fructose 1-6 bisphosphatase 1

catalyzes hydrolysis reaction of a phosphate

Dephosphorylation of fructose 1-6 bisphosphate

• the glycolytic reaction catalyzed by PFK-1 is exergonic and therefore irreversible, so it must be bypassed by adding water and breaking the phosphate bond to turn it into fructose-6-phosphate

• uses fructose 1,6 bisphosphatase-1 and Mg+

• irreversible

The third bypass reaction

dephosphorylation of Glucose-6-phosphate

Important fact of gluconeogenesis

it is energetically expensive but essential

dephosphorylation of Glucose-6-phosphate

• glycolytic reaction using hexokinase is highly exergonic and irreversible using ATP

• uses glucose-6-phsphatase and Mg+ to hydrolyze the phosphate bond and make glucose

• irreversible

why is it okay for gluconeogenesis to use so much ATP?

• some tissues need glucose

• there is still huge profit from glycolysis because gluconeogenesis does not actually use THAT much ATP

• to ensure irreversibility of gluconeogenesis

• to prevent waste and excretion of pyruvate

• many molecules feed into gluconeogenesis

What energetic molecules does gluconeogenesis use to make a full molecule of glucose?

• 4 ATP

• 2 GTP

• 2 NADH

coordinated regulation of glycolysis

• occurs in many tissues like the brain, liver, kidney, muscle

• provides ATP to support evergy-requiring processes

• net generation of 2 ATP, 1 NADH

coordinated regulation of gluconeogenesis

• occurs mainly in the liver

• generates glucose for export to other tissues in energy depleted conditions

• uses 4 ATP, 2 GDP, 2NADH

what happens during simultaneous glycolysis and gluconeogenesis?

ATP is broken down and heat is dissipated, wasting energy

how do we get glycolysis and gluconeogenesis to happen reciprocally?

• PFK-1 in glycolysis is activated by fructose 2,6 bisphosphate

• Fructose bisphosphatase in gluconeogenisis is inhibited by fructose 2,6 bisphosphate

• we can also do it physically

example pentoses

• ribose

• deoxyribose

glucose produced by gluconeogenesis in the liver/kidney is…

very quickly transported to other tissues by the bloodstream and the GLUT transporter and therefore not available for glycolysis in hepatocytes

physical barrier for reciprocal glycolysis and gluconeogenesis?

• glucose 6-phosphatase, the last enzyme in gluconeogenesis is present only in the ER membranes of the liber and kidney

• its active site faces the ER lumen

• the way hepatocytes separate glycolysis from gluconeogenesis

how is fructose 2,6 bisphosphate generated?

• PFK-2 makes it using ATP

• FBpase-2 breaks it

analogy for pfk-2 and FBPase-2

they are connected like sides of a coin

when the enzyme is not phosphorylated by phospho-protein phosphatase

only PFK-2 is active

When the enzyme is phosphorylated by camp-dependent protein kinase

FBPase-2 is active

glucagon

inhibits glycolysis

insulin

stimulates glycolysis

Pythagoras did not eat falafel

• fava beans contain a molecule that causes oxidative stress and red blood cells to lyse

• release of hemoglobin, jaundice, and kidney failure

• can be fatal, similar symptoms from ingestion of antimalarial drugs

oxidation of lipids

breaks membranes

pentose phosphate pathway

• generates NADPH and Ribose-5-phosphate

• rapidly dividing cells need more RNA, DNA, ATP, FADH, and NADH

• divided into oxidative and non-oxidative phases

what do we need when cells are going under rapid division?

they need pentoses from the pentose-phosphate pathway

NADPH is used to

counter oxidative damage in cells that synthesize fatty acids (liver and fatty acids) or RBC’s that carry oxygen

Step 1 Oxidative phase of pentose phosphate pathway

• start with glucose-6-phosphate

• OH attached to carbon number 1 is oxidized using G6P dehydrogenase into a carbonyl group

• making one NADPH and 6-Phosphoglucono-lactone

Step 2 oxidative phase of pentose phosphate pathway

• 6-phospho-glucono-lactone is turned into 6-phosphogluconate using lactonase

• turns the carbonyl keto group into a carboxylic acid group

Step 3 oxidative phase of pentose phosphate pathway

• 6-phosphogluconate is oxidized into ribulose-5-phosphate using 6-phosphogluconate dehydrogenase

• releases CO2 and makes NADPH

Step 4 oxidative phase of pentose phosphate pathway

• ribulose-5-phosphate is isomerized using phosphopentose isomerase into ribose-5-phosphate

• converts keto group into aldehyde

overall equation of oxidative phase

g6 phosphate + 2NADP+ + H20 → Ribose-5 phosphate + CO2 + 2NADPH + 2H+

Rapidly growing cells use…

the oxidative phase

Cells going through oxidative stress and need NADPH…

recycle the pentose sugars formed

goal for non-oxidative pentose phosphate pathway

convert 5 carbon ribose 6 phosphate back to 6 carbon glucose 6 phosphate

the non-oxidative phase of the pentose phosphate pathway part 1

• ribose 5 phosphate is isomerized into ribulose 5 phosphate using isomerase

• Ribulose 5- phosphate is epimerized into xylulose-5 phosphate

the non-oxidative phase of the pentose phosphate pathway part 2

xylulose 5 phosphate combines with a ribose molecule to form sedoheptulose-7phosphate and glyceraldehyde-3-phosphate using transkelotelase

the non-oxidative phase of the pentose phosphate pathway part 3

glyceraldehyde 3-phosphate and sedoheptulose-7phosphate are rearranged using transaldolase and form fructose-6 phosphate and erythrose-4-phosphate

the non-oxidative phase of the pentose phosphate pathway part 4

• fructose 6 phosphate is directly converted into glucose-6phosphate using phosphohexose isomerase

• erythrose-4 phosphate is combined with another xylulose 5 phosphate using transkelotalase to make fructose 6 phosphate and glyceraldehyde 3-phosphate

• the fructose-6 phosphate can be directly isomerized into G6P but the G3P uses triose phosphate isomerase, aldolase, and fructose 1,6 bisphosphatase to turn into F6P and then into G6P

which pathway does the fructose-6-phosphate and the g3p enter to turn into glucose-6-phosphate after the non-oxidative pentose phosphate pathway

gluconeogenesis

roadmap for the non-oxidative phase

5 molecules of 6 carbon sugars are made from 6 molecules of 5 carbon sugars

Epimerization of ribulose-5-phosphate

• occurs because the last reaction of the oxidative phase is reversible and the ribose can be turned back into ribulose

• is a readily reversible reaction that allows coexistence of the reactant and product

• uses ribulose-5-phosphate epimerase, and OH group gets flipped on the third carbon

epimer

differ in the orientation of groups bound to the chiral carbon atom

xylulose vs ribulose

epimers at the 3rd carbon position

when the 5 c molecules are making one 7 and one 3 c molecules what is happening?

reactions that make and break carbon-carbon bonds

what enzyme is unique to the pentose phosphate pathway?

transketolase and TPP

description of the first reaction in the roadmap, combination of ribose and xylulose into g3p and sedoheptulose

a transfer of a 2-carbon moiety from a ketose donor to aldose acceptor

TPP / thiamine pyrophosphate

• cofactor used by transketolase

• has a thiazollium ring

• ring resonance stabilizes the carbanion in the dihydroxymethyl group that is carried by trasketolase to make or break c-c bonds

importance of the thiazollium ring

• the nitrogen can exist with a positive (double bond) or negative charge (single bond)

• the carbon attached to the nitrogen is attached to the substrate

• positive nitrogen stabilizes negative charge on substrate

the second reaction catalyzed by transaldolase

• similar to aldolase from glycolysis

• protonated schiff base stabilizes the carbanion intermediate

• has a lysine residue with an amide group that can stabilize a carbanion

excess NADPH impact

feedback inhibition to the pentose phosphate pathway and more glucose is available for glycolysis

why wouldnt eat pythagoras eat falafel?

• G6P dehydrogenase deficiency prevents first reaction of the oxidative phase

• found in 25% of africa, middle east, and southeast asia

• protects against malaria which is good because they are susceptible to antimalarial drugs

• falafel causes oxidative stress

fava beans

have divicine which reacts with oxygen to make superoxide radicals that causes oxidative damage

which molecules cause oxidative stress

• mitochondrial respiration

• ionizing radiation

• sulfa drugs

• herbicides

• antimalarials

• divicine

why are superoxide molecules bad?

• they make hydrogen peroxide which is neutralized by glutathione peroxidase

2GSH meaning

glutathione with a reduced thiol group

glutathione peroxidase

neutralizes and reduces hydrogen peroxide into water and oxidizes 2gh into gssg

glutathione reductase

• regenerates glutathione peroxidase but uses NADPH in the process

Glycogen

• storage form of glucose in animals found in the liver or muscle as granules

• source of quick glucose and gets depleted in 24 hours

• repeating glucose with alpha 1-6 linkages which means branching

glycogen in muscles

beta granules ranging from 20-30 nm in diameter which each granule having 55k monomers

glycogen in liver

beta granules clump together to form alpha granules which can be 300 nm in diameter

overall structure of glycogen

• amylopectin

• linear and branched polymers of glucose

• the core is a dimer of the protein glycogenin

• tiered arrangement of chains provides many carbon 4 non-reducing ends facing outward for synthesis and breakdown for glycogen molecules

what catalyzes the first step in glycogenolysis

glycogen phosphorylase

starch is mostly

amylose

the first step in glycogenolysis

• Glycogen phosphorylase catalyzes the phosphorolysis of alpha 1-4 glycosidic linkage at the non-reducing ends of glycogen

• Pi attacks the non-reducing end of glycogen and removes terminal glucose residues as glucose-1-phosphate

• Repetitive action removes glucose-1-phosphate residues until 4 glucose residues are left on the chain before the branch point

phosphatase

removal of phosphate using water

phosphorylase

adds inorganic phosphate to break something