Biochemistry II Notes - Module 1

8/2/2019 Biochemistry II Notes - Module 1

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Lecture #1 (Wednesday, 1/18/12)

Generalized Metabolic Routes (i.e. cellular respiration):

1. Catabolic pathways- Breakdown of substrates.- Most catabolic reactions are oxidative (they remove electrons from substrate).- Energy is produced.- Provide building blocks for other macromolecules

2. Anabolic pathways- Biosynthesis: making more complex macromolecules.- Most anabolic reactions are reductive (electrons are added to substrate).- Energy is being utilized.

Catabolic enzymes 1/3 of

free energy is released

2/3 of E released

*Things to Review: Organic chemistry basics; redox reactions; thermodynamics.

3 stages of metabolism (see Figure 12.1 on next page)

Nutrient or

substrateAmphibolic intermediates

C02 + H20Complex molecules of the cell


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Lecture #2 (Friday, 1/20/12)

Are anabolic and catabolic pathways (e.g. glycolysis and gluconeogenesis) simply the reverse ofone another?

Answer: NO. If the same enzyme is used for two opposite processes, there would be no room for

regulation and activities would be futile.

Reasons:

1. The cell wouldnt simultaneously break down and synthesize glucose that would beredundant.

2. Also, a catabolic process is only favorable in one direction, where the overall G is negative(the process is exergonic).

Important Notes: ATP is the bodys predominant energy source. See Figure 3.7

ATP hydrolysis is favorable (energy is released when ATP is split) for these reasons:

1. Resonance of products2. Removal of products3. Repulsion is reduced4. Increase in entropy5. Most common pathway is ATP ADP + Pi. This is because the cells machinery is geared towards

synthesis from ADP rather than AMP.


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Figure 3.7

*Things to Review: basics of enzymes and how they are regulated (Chapter 11 - Enzymes: Biological

Catalysts; pp. 360-61, 403-7)

Enzymes are biological catalysts:

- mostly proteins (some RNA ribozymes)- lower activation energy (EA) of a reaction


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- have high specificity for substrates- work under mild conditions in the body- they can be regulated!!!

IMP: G does not change! Free energy of reactants and products stay the same. Enzymes do not

affect the thermodynamics of a reaction, only kinetics.

(See Handout 1 Fig. 2.1 Energy profile of a catalyzed and an uncatalyzed reaction.)

Lecture #3 (Monday, 1/23/12)

See Figure 11.15

*Things to Review: How does regulation affect the Km?

- Vmax: maximum rate of production. The enzyme is working as fast as it can.- KM: dissociation constant.- kcat: turnover number.- kcat/KM = efficiency


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There are 6 basic ways that enzymes are regulated. Ask yourself: what kind is this? Read pp. 398-407

A. Regulation of Enzymes 6 major metabolic control mechanisms.1. Substrate-level control

a. Concentration of S. Increase in [S] will result in increase of enzyme activity and vice versa.b. Concentration of P. Increase in [P] will result in decrease of enzyme activity negative

feedback.

Example:

Glucose + ATP --------------------> G6P + ADP

The accumulation of the product, glucose-6-phosphate, to a certain level will inhibit the activity of

hexokinase.

2. Compartmentation where is this process happening?This form of regulation has to do with the cell controlling the flow of substrates and products across

membrane.

3. Enzyme concentration regulation of synthesis and degradation. Synthesis of an enzyme can be regulated either at the level of transcription or translation.

transcription translation

Enzymes have half-lives. This another way that their concentration is controlled. They degradeafter a certain amount of time. See Handout 2 (Tabelle 2.1)

Ubiquitylation targets the enzyme to be digested in a lysosome. This is non-specific.

hexokinase

DNA RNA Protein


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Or, it can be specific in a lysosome or non-lysosomal degradation pathway. See Handout 3(Figure 2.15A or 2.7 Regulation of Enzyme Activity by Proteolysis). There are 4 E3 subtypes of

specific, non-lysosomal degradation pathways of enzyme regulation:

a. N-end rule E3 enzymes (in yeast) recognize basic or bulky/hydrophobic AA.b. Hect domain E3 enzymes (in mammals) active site Cys residue near the C-terminus.c. Cyclosome AKA, anaphase-promoting complex recognizes destruction box atthe N-terminus.

N-term-R-A-L-G-V-I-N-etc.

d. Phosphoprotein-ubiquitin ligase complex recognizes PEST sequence at the C-terminus.

* (Be sure to have a good table of Amino Acids AA that you like and can refer to easily)

4. Proteolysis zymogens, or pro-enzymes.These enzymes are inactive when synthesized, but are activated upon cleavage (by another enzyme). The

activation of zymogens involves a conformational change in the enzyme and exposure of its active site.

Enzyme Enzyme*

See Figure 11.39

Red arrow = autocatalytic


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5. Covalent modifications methylation, acetylation, phosphorylation, etc.

Covalent mods of enzymes can work either to activate an enzyme or to inhibit its function. This method of regulation is usually irreversible. Kinases phosphorylate. Phosphatases dephosphorylate.

(Lecture #4 (Wednesday, 1/25/12 Enzyme Regulation continued)

6.

Allosteric effectors the presence of certain molecules can affect enzyme activity.

Low molecular weight organic compounds Proteins Metal ions NOT the immediate substrate or product of the enzyme.

(*Note: If an enzyme is inhibited by its immediate product, then this is substrate-level control.)

Enzymes controlled in this fashion tend to have multiple subunits with multiple active sites. Theyexhibitcooperative substrate binding.

Q: How do we know?

A: Shape of kinetics curve!

See Figure 11.32 on the next page.


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Figure 11.33

[S]c is known as the critical substrate level. At this concentration, a small change in [S] results in avery large change in V!

Thus, the cell can keep [E] within a very narrow range. COOPERATIVITY IS COOL!!!These enzymes exhibit two binding states, tense (T) and relaxed (R):

TR

Low affinity

for S

High KM

High affinity

for S

Low KM

Effector

Molecules


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(+) effector molecules = activators stabilize R state

(-) effector molecules = inhibitors stabilize T state

Figure 11.34 We see this a lot in metabolism

Activator will lower KM and shift the curve to the left V is increased. Inhibitor will raise KM and shift the curve to the left V is decreased. Always ask, WHAT IS GOING ON WITH THE KM???


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Metabolism of Sugar

I. Anaerobic ProcessesCarbohydrate metabolism was the first pathway to be well defined.

Glycolysis sweet-splitting Gluconeogenesis making sweetsA. GLYCOLYSIS

See Figure 13.2 Big picture.


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Glucose + 2ATP 2 ATP + 2 NADH + 2 Pyruvate

(Lecture #5 (Friday, 1/27/12 Glycolysis continued)

Glycolysis occurs in the cytosol of the cell. IMPORTANT: Make sure that you are able to track labeled carbons throughout a pathway. Also, make sure to review your organic chemistry.

See:

- Overview of Glycolysis- Handout 4 The Glycolytic Pathway- Figure 13.3 An Overview of Glycolysis- Handouts 5 and 6 high-energy compounds

There are 10 steps in Glycolysis, each mediated by 10 different enzymes, respectively. These are in turn

grouped into the (1) ATP Investment Phase and the (2) Energy Generation Phase:

Phase (1)

1. Hexokinase phosphorylates glucose to produce glucose-6-phosphate.2. Isomerization of glucose-6-phosphate to fructose-6-phosphate by phosphoglucose isomerase.3. Phosphorylation of F6P to fructose-1, 6-bisphosphate by phosphofructokinase.4. Phosphorylation and cleavage of FBP to glyceraldehyde-3-phosphate and dihydroxyacetone-

phosphate by aldolase.

5. Isomerization of DHAP to G3P by triose phosphate isomerase.Phase (2)

6. Oxidation and phosphorylation of G3P to 1, 3-bisphosphoglycerate by G3P dehydrogenase.7. Substrate-level phosphorylation of 1, 3-BPG to 3-phosphoglycerate by phosphoglycerate kinase.8. Isomerization of 3-phosphoglycerate to 2-phosphoglycerate by phosphoglycerate mutase.9. Dehydration of 2-phosphoglycerate to phosphoenolpyruvate by enolase.10.Substrate-level phosphorylation of phosphoenolpyruvate to pyruvate by pyruvate kinase.

See Diagrams/Flowcharts of Glycolysis.


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Regulation of Glycolysis

Important enzymes involved in the regulation of glycolysis are insulin and glucagon. Overview: Glucose Pyruvate. However, pyruvate has many different pathways that it may enter.

See Handout 7 Figure 13.14 Metabolic fates of pyruvate.

See Handout 8 Figure 7.4 (Glycolysis 271) to see how glucose is metabolized in different cells.

The liver is the only tissue that can also exportglucose.Why is this? This is because the liver is the main organ that controls or regulates the amount of sugar in

the blood.

In the liver, there is no hexokinase, but glucokinase. This is important! Glucokinase has a higherKM so that it doesnt consume all the sugar, but is able to send itvia the bloodstream to the brain if

needed. The brain is pretty picky it only uses glucose for energy.

The normal concentration of glucose in the blood is about 5mM.

See Handout 9 Figure 7.13 Important regulatory features of the glycolytic pathway

See Handout 10 Figure 14.14 Comparison of the substrate saturation curves for hexokinase and

glucokinase.


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(Refer back to Handouts 9 and 10)

There is a certain pattern that we see in regulation of metabolism:

The first unique step is usually the one that is highly regulated.For example, in glycolysis, steps 1 and especially 3 are regulated. Step 3 involves the production of

fructose-1, 6-bisphosphate, which doesnt branch off to other pathwaysits unique to glycolysis.

Steps of Glycolysis that are regulated:

Step 1 (Hexokinase phosphorylates glucose to produce glucose-6-phosphate) is regulated by:

Hexokinase is inhibited by an increased concentration of glucose-6-phosphate. This would beknown as substrate-level regulation of enzyme activity.

Step 3 (Phosphorylation of F6P to fructose-1, 6-bisphosphate by phosphofructokinase) is regulated by:

Activated (+) AMP, fructose-2, 6-bisphosphate Inhibited (-) ATP, citrate, H+

See Figure 13.9 shows how certain effector molecules affect step 3, or specifically, the KM of PFK.

Step 10 (Substrate-level phosphorylation of phosphoenolpyruvate to pyruvate by pyruvate kinase) is

regulated by:

Activated (+) Fructose-1, 6-bisphosphate. For two reasons:- First unique product of glycolysis- Ensures that the glycolysis reactions go to completion. Makes sure that the final product (pyruvate

is achieved) and thus pull all of the in-between reactions follow through to the end.


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Figure 13.9

(Also, refer to Handout 9 again at this point)

*Think about exactly why these molecules either activate or inhibit glycolysis. For example, why would

AMP activate step 3 of glycolysis? This is because a high concentration of AMP would indicate a state of

ATP shortage (energy shortage) for the cell and would signal the cell that it needs to make more ATP by

breaking up some sugar for energy!!

See Handout 11 Figure 14.19 Overview of mechanism for glucagon inhibition of hepatic glycolysis.


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See Handout 12 specific diagram of how glucagon decreases the production of F-2, 6-BP.

Glucagon

This enzyme is released when blood sugar is low. Glucagon causes a decrease in fructose-2, 6-

bisphosphate.

Q: What does this do?

A: It inhibits glycolysis.

See Handout 12

Again, think about exactly how this happens. Since fructose-2, 6-bisphosphate is a strong activator of step

3 of glycolysis, if its production were hindered, then glycolysis would be inhibited.

Insulin

This enzyme is released when blood sugar gets too high.

Insulin activates cAMP phosphodiesterase, which causes the breakdown of cAMP. This countersthe effect of glucagon, which activates the production of cAMP.

Also, insulin activates phosphoprotein phosphatase, which works to increase the production offructose-2, 6-bisphosphotase, a strong activator of step 3 of glycolysis.

See Handout 12 to see how all this happens.


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Q: Why would insulin work to counter the effects of glucagon and activate glycolysis?

A: If there is too much sugar in the blood, it would make sense to produce insulin hormone to tell cells to

start breaking down this excess sugar (glycolysis) and also, in turn, stop the effects of glucagon, which is a

hormone that is telling cells to stop breaking down sugar and actually make more.

Lecture #8 (Friday, 2/3/12)

Gluconeogenesis Chapter 16

Whereas glycolysis is a catabolic process (energy is released) whereby we obtain energy,

gluconeogenesis is an anabolic process whereby we store energy in the form of glucose.

Read pages 415-18 about anabolism.

Glycogen = chain of glucose

See Figure 16.2 on the next page.


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The Brain and the CNS and PNS use glucose as their primary source of energy. Muscle tissue uses glucose as its primary energy source as well.

Glucose consumption per day:

Overall 160 grams

Brain 120 grams

This means the brain consumes 120/160, or 75% of the glucose that our body uses in a day!

Our body can store about 190 grams of glucose, or about a days worth of good old glucose. So, sugar is importantits not a bad thing, unless it is in excess.


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Gluconeogenesis is the process whereby our bodies make sugar, or glucose.

Substrates (carbon sources) for making glucose:

1. Amino acids (dietary)2. Propionate (from fatty acids)3. Lactate (from pyruvate)4. Glycerol (from fats)

See Figure 16.4


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What do we do with the sugar that we make?

1. Use it.2. Store it.3. Glycoproteins.4. Glycolipids.

See Figure 16.3 on the next page overview of the energy consumed to make glucose (gluconeogenesis).

Also, note that there are new enzymes that are specific to gluconeogenesis and act contrary to the steps of

glycolysis. This makes sense because these are essentially opposite processes. New enzymes:

- Pyruvate carboxylase at step 10 of glycolysis.- PEPCK at step 9.- Fructose-1, 6-bisphosphatase at step 3.- Glucose-1, 6-bisphosphatase at step 1.

These are all enzymes specific to gluconeogenesis that are needed to make glucose from pyruvate(or another carbon source, listed above and shown in Figure 16.4).

However, the energy required to make glucose (the ATPs, GTPs, NADHs) comes from fatty acidcatabolism.

The biggest carbon source for gluconeogenesis is lactate See Figure 16.5 For some reason, we cant use leucine or lysine as carbon sources for gluconeogenesis.


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Figure 16.5


Regulation of Gluconeogenesis

*Read pages 568-72

Effector molecules that activate glycolysis may inhibit gluconeogenesis, and vice versa. This is because they are essentially opposite processes and consequently very interdependent.

See Figure 16.3 again.


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1. Overview In glycolysis, steps 1, 3, and 10 are heavily regulated For gluconeogenesis, the body uses specific enzymes to ensure completion.

*IMPORTANT: Be comfortable tracking labeled carbons through glycolysis and gluconeogenesis,

respectively (See Question 3 from the old exam).

We need energy for gluconeogenesis (anabolism) we get it from fat! Fat is broken down into 3 fatty acid chains and 1 glycerol molecule. Fatty acids are converted into

acetyl CoA and feed into the TCA cycle.

2. Fructose-2, 6-bisphosphate- Activates glycolysis- Inhibits gluconeogenesis

3. Hormonal Control Regulating the supply of fatty acids to the liver. Regulating the supply of enzymes for either glycolysis or gluconeogenesis.


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See Figure 16.6 Side by side comparison of regulation in glycolysis and gluconeogenesis.

*See Dr. Ks notes on molecules and conditions that regulated glycolysis and gluconeogenesis.


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(a)Glucagon sends a message to make more glucose (dont break it down). i. Raises the concentration of free plasma fatty acids this is accomplished by raising the level of

lipolysis in adipose tissue.

ii. Raises cAMP levels in the liver. Here are some functions of cAMPa) Activates protein kinase that phosphorylates PFK-2. This phosphorylated PFK-1 is now a

phosphatase that converts F-2, 6-BP to F6P. This helps to inhibit glycolysis (See Handout

12)

b) Stimulates the breakdown of glycogen. This makes sense because we need more sugar inthe blood.

c) Activates protein kinase A. This enzyme deactivates pyruvate kinase by phosphorylating it,which inhibits step 10 of glycolysis.

iii. Activates transcription of PEPCK gene.iv. Induces the synthesis of fructose-1, 6-bisphosphatasev. Induces the synthesis of glucose-6-phosphatasevi. Represses the synthesis of these enzymes involved in glycolysis (thus inhibiting it):

a) Pyruvate kinaseb) PFKc) Glucokinase

(b)Insulin sends a message to start breaking down glucose because theres too much in the blood. i.

Inhibits the transcription of PEPCK

ii. Represses the synthesis of fructose-1, 6-bisphosphataseiii. Represses the synthesis of glucose-6-phosphatase


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Glycogen Biosynthesis and Breakdown

*Read pp. 572-78

Overview

1. Glycogen is a stored form of glucose Glycogenolysis breakdown of glycogen. Occurs in the liver. Glycogenesis synthesis of glycogen. Occurs in the muscle.

Muscle 1-2% of its wet weight is glycogen.

Liver 10% of its wet weight is glycogen.

Overall, however, we have more glycogen in our muscle tissue because we have lots of muscle andonly one liver.

2. Glycogen phosphorylase catalyzes the first step in glycogen degradation.

See Glycogenolysis Handouts Figure 7.52 and 7.54

3. Synthesis of glycogen requires unique enzymes.

See Figure 16.8


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Glycogen primer = Primed glycogenin

See Handout Figure 7.56


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Glycogenolysis (breakdown)

Glycogen Glucose-1-phosphate

UDP-Glucose monomer Glycogen

*Remember to always look at the big picture! How does glucagon or insulin affect each of the four

pathways we studied?

Imagine your blood sugar is low. Your body recognizes this and releases glucagon to raise yourblood glucose level because your brain needs food!!

Glucagon [Glucose] in blood goes up

(-) Glycolysis (+) Gluconeogenesis (+) Glycogen breakdown (-) Glycogen synthesis

See Figure 16.11 on the next page. This happens in the liver

l co en hos hor lase

l co en s nthase


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Figure 16.12 2C also inhibits PP-1 so that synthase is not activated and phosphorylase is not inhibited.

Imagine that blood glucose levels are low. Your body recognizes this and releases insulin to loweryour blood glucose level because too much sugar in the blood is bad!

Insulin [Glucose] in blood goes down

(+) Glycolysis (-) Gluconeogenesis (-) Glycogen breakdown (+) Glycogen synthesis

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Biochemistry II Notes - Module 1

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