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number 11

Done by حسام أبو عوض

Corrected by Moayyad Al-Shafei

Doctor Nayef Karadsheh

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General Regulatory Aspects in Metabolism:

We can divide all pathways in metabolism to catabolicand anabolic. In

catabolic pathways dietary molecules are broken down to produce energy-rich

molecules (ATP) and also generate NADH (mainly). Then in anabolic pathways

this energy is used to build upour macromolecules (proteins, fats, etc.).

-Catabolic reactions end by producing energy-poor products (CO2, H2O, NH3).

- NADH is produced in catabolic reactions and is later used in the electron

transport chain to obtain energy by converting NADH back to NAD+ ,whereas

the role of NADPH is mostly anabolic reactions, where NADPH is needed as a

reducing agent .

Cells only use the required amounts of resources (no excess). To do such a job,

enzymes with regulatory subunits (in addition to the catalytic subunit) are

needed. Signals can regulate the activity of such enzymes.

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The mechanisms of regulation can vary, they can be :.

- Signals from within the cell :.Intracellular communications typically lead to

rapid responses.

-Having cells exchange substances with each other via cell junctions :. Gap

junctions allow direct communication between adjacent cells (see

introduction to histology).

-Using the second messenger system.

The two most common second messenger systems are the calcium

phosphatidylinositol system and the adenylyl cyclase system (the latter used

in carbohydrates and fats metabolism).

[Example on adenylyl cyclase system] A hormonal signal (e.g glucagon) binds to

the extracellular part of a receptor on the membrane of the cell. This binding is

transmitted across a domain within the cell membrane and then to the

intracellular part

of the receptor.

The receptor

would be coupled

to a G-protein.

The signal could

stimulate or

inhibit the G-

protein. The G-

protein consists

of 3 subunits, α, β

and ϒ. Before the

signal a GDP

molecule would

be bound to the α

subunit. Upon

binding, a GTP molecule would replace the GDP molecule which activates the

α subunit letting it dissociate from the α, β and ϒ complex. The α subunit then

interacts with another membrane bound enzyme, adenylyl cyclase. Adenylyl

cyclase begins converting ATP into cyclic AMP and this continues as long as

the first signaling molecule remains attached to the receptor. When the

signaling molecule leaves,the GTPase enzyme present in the α subunit changes

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GTP to GDP,sodeactivating the α subunit and reuniting it with the β and ϒ

subunits.

The produced cAMP (cyclic AMP) binds to cAMP dependentprotein kinase

enzyme in the cytosol. The protein kinase is formed from four subunits (2

regulatory and 2 catalytic). Upon cAMP binding, the regulatory subunits

dissociate and the enzyme becomes active. This enzyme phosphorylates many

other enzymes in the cell and this phosphorylation activates some enzymes

and inhibit others (also can work on ion channels and DNA promoter regions

[increases transcription]).

These changes that occurred in the cell must be reversed after some time for the

cell to maintain its physiological state. Protein phosphatase enzyme is

activated and removes the phosphates from the other enzymes returning the cell

to its original status.

Phosphodiesterase enzyme converts cAMP to 5’-AMP (stopping the action of

cAMP)

Glycolysis

It is a universal pathway used by all cell types and by all organisms (aerobic and

anaerobic) in order to generate ATP.In addition to that, it provides the

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precursors for anabolic pathways; for example, some amino acids are

produced from its intermediates, in addition to fats.

-Glycolysis can be divided into 2 main phases…

1- Preparative phase (energy-investment phase):. This phase consumes 2

ATP molecules in order to phosphorylate glucose (energizing it).

2- ATP generating phase:. This phase includes 2 NADH molecules and 4

ATP molecules production. So glucose will be converted to 2 molecules

of pyruvate by the end of this phase.

-Under anaerobic state only 2 ATP are formed at substrate level with

conversion of glucose to lactate.

-Under aerobic state glucose converted to CO2 (oxidized completely)and

approximately 38 ATP molecules .

At the end of the process (10 steps) pyruvate is produced. This can then go to

an anaerobic pathway (2 ATP produced) or an aerobic pathway (36-38 ATP

produced).

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Some tissues have an absolute requirement of glucose, this includes: brain,

RBC’s, eyes (cornea lens and retina), kidney medulla, testes, leukocytes and

white muscle fibers (in excessive exercise it will undergo both aerobic and

anaerobic respiration).

Steps of Glycolysis

Step 1

Glucose enters the cell and is immediately phosphorylatedto glucose-6-

phosphate, this reaction requires the use of an ATP molecule. The reaction is

catalyzed by two isoenzymes (according to position), glucokinase (Liver and

beta cells) and hexokinase (most of the cells).

-This reactionis also the first one in glycogenesis andpentose-

phosphatepathway .

-Glucokinase has a higher Km and a higher Vmax (refer to summer

biochemistry course) than hexokinase. This means that glucokinase has a

lower affinity to glucose but can take more of it. This is completely suitable

with its role in the liver, for when glucose concentration in the blood increases

(above 100mg/100mL) the glucokinase enzyme will operate and phosphorylate

glucose for storage (and if the storage gets full, glucose is changed to fat), but

if the glucose concentration drops glucokinase will not function. Hexokinase,

on the other hand, will always operate whether the concentration of glucose in

the blood is high or low, this is due to its high affinity to glucose and this suits

its role in supplying cells with glucose for energy production, cells must always

get their energy requirement regardless of the situation.

-Both hexokinase and glucokinase work best with glucose but can also work on

other sugars like fructose, mannose and galactose.

-Glucokinase’s action is induced by insulin in hyperglycemia, hexokinase’s is

never induced.

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-During fasting glucose concentration in blood drops and the liver does not

take any glucose, in fact it releases glucose to the blood stream from its

storage.

-This reaction is essentially irreversible (exergonic) and hexokinase is one of

the regulated enzymes in glycolysis.

Step 2

Phosphoglucoseisomerase (aldose-ketose isomerization) enzyme changes

glucose-6-phosphate to fructose-6-phosphate. This is a reversible reaction.

Step3

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Phosphofructokinase enzymecatalyzes the use of ATP to convert fructose-6-

phosphate to fructose-1,6-bisphosphate. This enzyme is the most important

enzyme in the regulation of glycolysis.

Step 4

Fructose-1,6-bisphosphate is cleavedinto the two trioses

dihydroxyacetonephosphate (a keto sugar) and glyceraldehyde-3-phosphate

(an aldo-sugar). This reaction is catalyzed by the enzyme aldolase.

Step 5

The two trioses produced are interconverted by the enzyme phosphotriose

isomerase. But, given that glyceraldehyde-3-phosphate is the one that

continues the pathway, overall, dihydroxyacetone-3-phosphate is the one

being changed to glyceraladehyde-3-phosphate. So, we get two

glyceraldehyde-3-phosphate molecules from each glucose molecule.

(from step 6 to 10 are the “oxidative steps”)

Step 6

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Glyceraldehyde-3-phosphate is converted to 1,3-bisphosphoglycerate via the

enzyme glyceraldehyde-3-phosphate dehydrogenase(GA3PD). The reaction

produces an NADH molecule.

Via a cysteine group in the active site, the enzyme forms a linkage with the

substrate. The enzyme has an NAD+ molecule close to the active site. Then the

substrate is oxidized forming a thioester linkage and NAD+ is changed to

NADH. NADH has a low affinity to the enzyme so it leaves and is replaced by a

new NAD+ molecule. An inorganic phosphate then attacks the thioester linkage

releasing the substrate as the product, 1,3-bisphosphoglycerate, and returning

the enzyme to its original form.

Step 7

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Phosphoglycerate kinase enzyme changes 1,3-bisphosphoglycerate to3-

phosphoglycerateand an ATP molecule. (Remember that from step 5 two

molecules form from each product, so we actually get two ATP molecules

here).

Step 8

Phosphoglycerate mutase enzyme (mutases change the position of a

functional group from one carbon to another) change3-phosphoglycerate to 2-

phosphoglycerate. The molecule 2,3-bisphosphoglycerate is needed in addition

to the enzyme for this reaction to occur (this is the only reaction in the cell in

which this molecule is used, so it is only seen in small amounts here. This

molecule is seen in large amounts in the blood where it regulates the red blood

cells).

Step 9

Enolase enzyme changes 2-phosphoglycerate to the high energy molecule

phosphoenolpyruvate. This happens through a dehydration (H₂O is lost)

reaction and a rearrangement reaction.

Step 10

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Phosphoenolpyruvate is converted into pyruvate using pyruvate

kinaseenzyme. An ATP molecule is produced in the process. (Remember it is

actually two ATP molecules produced by one molecule of glucose).

This reaction is irreversible and this enzyme is among the ones that are

regulated. (Notice that all the three regulated enzymes are kinases).

The production of ATP in glycolysis is called as substrate level phosphorylation

(like Krebs cycle).

Metabolic Fates of Pyruvate

Anaerobic Respiration

Mammals

Pyruvate is changed to lactate using the lactate dehydrogenase enzyme which

allows us to reconvert NADH to NAD+ for it to be used in converting more

glucose to pyruvate and producing more energy. This is seen in muscle fibers

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during intense exercise and in the red blood cells even with no exercise simply

because there is limited oxygen compared to the amounts of pyruvate. This

causes accumulation of lactate, so this lactate moves through the blood to the

liver where it is changed back to pyruvate and back to glucose to be used again

by the muscles to produce more energy. The liver oxidizes fat in order to get

energy for formation of glucose from lactate.

(The build-up of lactate causes fatigue).

Yeast (and Some Bacteria)

These follow the alcoholic system. Here pyruvate is changed via pyruvate

decarboxylase (Thiamine pyrophosphate (TPP) is needed) into acetaldehyde

and CO₂ (which is excreted). Acetaldehyde is changed to alcohol via alcohol

dehydrogenase enzyme and in the process NADH is converted back to NAD+

to be used again in glycolysis. (No alcohol is produced in humans because they

do not have pyruvate decarboxylase).

When ethanol reaches 10-11% in concentration it begins affecting the

efficiency of the bacteria, just like lactate causes fatigue to us. (Alcohol

companies have different methods to increase the concentration of alcohol).

Alcoholic fermentation is also used in home. In baking bread when yeast is

added to the dough partial alcoholic fermentation occurs and CO₂ forms raising

the dough. When the dough is placed in the oven the baking removes all the

alcohol (evaporates it) so the smell of bread is actually that of the evaporated

alcohol.

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Aerobic Respiration

Pyruvate is changed to acetyl CoA via the pyruvate dehydrogenase complex.

When acetyl CoA is in excess of the requirement of Krebs cycle the excess will

be changed to fat.

When fasting amino acids can be used to make pyruvate (due to shortage of

sugar) then pyruvate is changed in the liver to glucose to maintain blood sugar

level. This glucose is NOT used in glycolysis. Instead, the required acetyl CoA is

produced from fat hydrolysis. Then this acetyl CoA will inhibit pyruvate

dehydrogenase complex to prevent wasting pyruvate in formation of acetyl

CoA and get pyruvate to be changed to oxaloacetate in order to be used in

both Krebs cycle and glucose synthesis.