Biochem Lecture 5

Exam 2 Content

What is a proteoglycan?

It is another type of protein with sugars attached to it, but it is made up of much more carbohydrates than glycoproteins are. Carbohydrates make up a larger part of proteoglycans than they do in glycoproteins.

• Proteoglycans consist of a protein core with multiple sugar chains. These sugar chains are called glucosaminoglycans (GAGs). 

• These GAGs are characteristic of proteoglycans (like how N-links and O-links are characteristic of glycoproteins). 

• They are used in connective tissue structure, lubrication, cell adhesion, and cell proliferation. 

Describe the structure of glycosaminoglycans

These sugar chains are made up of repeating disaccharides of a derivative of an amino sugar like glucosamine or galactosamine (meaning glucose with an amine or galactose with an amine). At least one of the sugars in the disaccharide with be an amino sugar. 

• At least one of the two sugars in the disaccharide will have a negatively charged carboxylate or sulfate group. 

  • These negative charges make GAGs very polar, which makes them useful for binding to polar molecules like water. 

Describe the structures of chondroitin-6-sulfate and keratan sulfate 

Both of these molecules are examples of glycosaminoglycans that can be apart of proteoglycans. 

Chondroitin 6-sulfate:

• Is made up of a glucose derivative linked to a galactose derivative via a β-1,3 linkage. 

• The glucose derivative is a sugar acid— gluconic acid. 

• The galactose derivative has an amine group that is acetylated and a sulfate group— N-acetylgalactosamine-6-sulfate. 

• Disaccaharides units are connected via β-1,4 linkages. Thus, chondroitin 6-sulfate molecules are connected to each other via these linkages. 

Keratan sulfate:

• Is made up of galactose linked to a glucose derivative via a β-1,4 linkage. 

• Galactose is not modified in any way. 

• The glucose derivative has an amino group attached to it that is acetylated and a sulfate group on carbon 6— N-acetylglucosamine-6-sulfate. 

• Disaccharides units are connected to each other via β-1,3 linkages. 

What is aggrecan? 

It is an example of a proteoglycan— a protein core with chains of sugar molecules (which are glycosaminoglycans).

• Each aggrecan is made up of 3 protein domains (G1, G2, G3) with the glycosaminoglycans between G2 and G3.

• Its protein core/domains is made up of 2397 amino acids. 

• Its glycosaminoglycans are made up of chondroitin sulfate and keratan sulfate. 

  • Water molecules bind to the charged groups (like sulfates and carboxylates) of the glycosaminoglycans. This makes them very hydrophilic.

• Many aggrecan molecules can be linked together by the protein hyaluronan— aka hyaluronic acid.

  • Thus, because the glycosaminoglycan chains have those charged groups that are hydrophilic and attract water between them, the protein hyluronon— hyaluronic acid— is very hydrophilic. 

Describe the relationship between human blood types and oligosaccharides

Red blood cells have specific carbohydrates (oligosaccharides) attached to their surface that determine the blood group that an individual has— A, B, or O. 

• Everyone is born with the oligosaccharides that makes up the O-antigen. This O-antigen is considered the core/foundation that all oligosaccharides on red blood cells have.

  • The O core is made up of galactose, GlcNAc, another galactose, and fucose.

• However, we can inherit glycosyltransferases— proteins with sugars attached to them— that will add specific sugars to the O antigen to make it an A, B, or both antigen.

  • Thus, our blood types are determined by the glycosyltransferases that we inherit from our parents because each glycosyltransferase only yields one specific oligosaccharide antigen.

  • We could inherit 0, 1, or 2 of the glycosyltransferases. 

  • The A-antigen has an extra N-acetylgalactosamine (GalNAc) added to the O antigen core via an α-1,3 linkage.

  • The B-antigen has an extra galactose attached to the O antigen core via an α-1,3 linkage. 

What are glycan-binding proteins?

Proteins that recognize and bind to specific carbohydrate structures— glycans— on other molecules. They facilitate cell-to-cell contact.

Explain hemagglutinin and neuraminidase

Hemagglutinin is a glycan-binding protein that exists on the surface of a viruses’ envelope.

• Once Influenza enters the human body, hemagglutinin binds to sialic acid, which is located on sialic acid receptors on host cells. Sialic acid is an acid sugar that is located at the ends of oligosaccharide chains that are present on glycoproteins and glycolipids on the cell surface.

• Once it binds, the virus enters the host cell, replicates its DNA and makes viral proteins and particles (virions) that are released once the host cell dies and go on to infect other cells in the body. 

• However, these new virus particles are still linked to the sialic acid receptors. For these particles to be released from the host cell and infect other cells, the link between hemaglutinin and the sailic acid receptor must be broken. 

• A viral enzyme called neuraminidase cleaves the oligosaccharide from the sialic acid receptor and thus allows the viral particles to be released from the host cell. 

  • Anti-flu medications like Tamiflu inhibit neuraminidase, which keeps hemagglutinin attached to sialic acid receptors and thus prevents viral particles from being released from the host cell and thus contains the virus. These medications should be taken when you first feel symptoms so that the virus can be contained early.

What is the purpose of glycolysis? 

Glycolysis is the first step of cellular respiration and it breaks down glucose to pyruvate. Thus, glycolysis is the metabolism of glucose to pyruvate. 

What are the 3 main parts of glycolysis?

1. The loading phase— is when glucose is phosphorylated and then isomerization occurs. 

2. Breaking up the 6 carbon molecule to two 3 carbon molecules

3. Metabolism of G3P to make 2 molecules of ATP

Describe the steps of phase 1 of glycolysis

This is the loading phase, and during these steps, glucose will be converted to fructose-1,6-biphosphate. 

1. Glucose enters the cell and is then phosphorylated by hexokinase as long as ATP is also present, which yields glucose-6-phosphate (which cannot be exported out of the cell). The phosphate group is attached to carbon 6. 

   • This step is thermodynamically irreversible— formation of products is favored because they are lower in free energy. Thus, the reaction occurs spontaneously in the presence of ATP. 

2. Glucose-6-phosphate is isomerized by phosphoglucose isomerase to form fructose-6-phosphate. Thus, an aldehyde sugar is rearranged to form a ketone sugar (aldose to ketose isomerization). 

   • Phosphate group does not move— it remains attached to carbon 6. 

   • Reaction is reversible— so fructose-6-phosphate could be isomerized by phospoglucose isomerase to reform glucose-6-phosphate. G-6P is in equilibrium with its open-chain form, F-6P’s open-chain form, and F-6P.

3. Fructose-6-phosphate is phosphorylated by phosphofructokinase (PFK) in the presence of ATP to form fructose-1,6-biphosphate (F-1,6-BP). Thus, carbon 1 gains a phosphate group.

   • This reaction is thermodynamically irreversible— formation of products is spontaneous and favored.

What do kinases do?

They phosphorylate molecules— meaning they add phosphate groups to substrates using ATP. They use this ATP for both energy and a phosphate group. 

Remember—kinases always need ATP for both the phosphate group and as a source of energy.

Note that the words that come with kinase indicate what it is phosphorylating. 

Example— hexokinase: phosphorylating a six-carbon sugar (like glucose or galactose)

Explain the relationship between the name of an enzyme and the relative stability of products vs reactants

The name of the enzyme indicates which molecule is more stable— the reactant or the product.

Example— glucose-6-phosphate is more thermodynamically stable than fructose-6-phosphate, hence the name of the enzyme and the fact that the reaction is reversible (it would want to go back to that more thermodynamically stable molecule).

Describe the steps in phase 2 of glycolysis

Fructose-1,6-biphosphate will be split into GAP and DHAP.

1. The enzyme aldolase breaks the bond between C3 and C4 of fructose-1,6-biphosphate, which splits the molecule in half and forms two molecules: dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP or G3P), each of which is a 3-carbon sugar (triose phosphates). Note that this split is reversible.

   • DHAP is a 3-carbon ketone sugar, and corresponds to the top half of F-1,6-BP (carbons 1-3). 

     • DHAP does not proceed through glycolysis. Rather, it is isomerized to GAP via triose phosphate isomerase. Now that it has been converted to GAP, it can proceed through glycolysis. 

     • Note that the isomerization with triose phosphate isomerase is reversible. It is a ketose to aldose isomerization. 

   • GAP is a 3-carbon aldehyde sugar and proceeds through glycolysis without any changes right now. GAP corresponds to the bottom of F-1,6-BP, thus its carbons are numbered 4-6 in relation to the carbons from its parent molecule. 

   • Thus, after this step, we have two GAP molecules from 1 molecule of glucose.

   • Note that all of the reactions in this step are reversible, and GAP could go all the way back to fructose-1,6-bisphosphate.

Naming of GAP and numbering

GAP recall is glyceraldehyde 3-phosphate. The phosphate group is attached to carbon 3 based on the numbering of the triose itself without comparing it to anything else. 

The numbering from glucose is not related to the numbering that relates to the location of the phosphate group. 

Recall that isomerases move functional groups within the same molecule to create a new isomer. Triose phosphate isomerase moves the carbonyl of C2 to C3, and so the OH from C3 must go to C2— carbonyl and OH switch spots, and that forms GAP. 

Because we get GAP from F-1,6-BP and isomerization of DHAP, the numberings of the carbons relative to the original F-1,6-BP molecule can be different. The numbering depends on whether it came from the initial production of GAP or from isomerization of DHAP. We cannot tell where each GAP molecule came from, so just note the two possibilities that each carbon can be. 

• The top carbon— the carbonyl— can be C3 or C4

• The second carbon can be C2 or C5

• The third carbon— with the phosphate group— can be C1 or C6

Give an overview of the steps in phase 3 of glycolysis 

The 2 GAP molecules will be converted to pyruvate through a two-step process.

1. GAP is converted to 1,3-biphosphoglycerate (2 of them because 1 for each GAP that enters) by glyceraldehyde-3-phosphate-dehydrogenase in a two-step process:

   1. Oxidation

   2. Phosphorylation

2.  

Describe what occurs during the oxidation step of the step 1 of phase 3 

• GAP is oxidized, meaning it loses electrons and gains a bond to oxygen. The H the carbonyl is bound to becomes an OH after oxidation. This forms 3-phosphoglyceric acid (glycerate is deprotonated) because water is deprotonated and OH- attacks the carbonyl carbon.

• NAD+ is also reduced to NADH because the it gained the electrons from the broken C-H bond. This leaves just H+.

• Thus, NAD+ is a cofactor of glyceraldehyde-3-phosphate-dehydrogenase and it must be replenished for glycolysis to occur (it is a limited quantity in the cell). 

• Thus, this step requires NAD+ as a cofactor and also needs H₂O to provide the OH that will be added to form 3-phosphoglycerate. NADH is also produced through reduction of NAD+ and H+ is released (from the C-H bond from carbonyl that is broken).

• This step is reversible

Note that this is the theoretical mechanism. This mechanism does not actually occur because 3-phosphoglyceric acid is very low in free energy (lower than G3P and 1,3-biphosphospglycerate), so glyceraldehyde-3-phosphate dehydrogenase, which uses inorganic phosphate not ATP, could not possibly phosphorylate such a low free energy molecule without an input of energy (from ATP, which it does not use). It would be non-spontaneous and would not occur. 

Describe what occurs during the phosphorylation step of step 1 of phase 3 

• 3-phosphoglyceric acid gets phosphorylated by glyceraldehyde-3-phosphate dehydrogenase and an inorganic phosphate.

  • This inorganic phosphate (has a lot of energy)— specifically its negatively-charged oxygens— attacks the carbon and replaces the OH attached to the carbonyl carbon.

  • H2O is released as a product

  • This forms 1,3-biphosphoglycerate

  • This reaction is very unfavorable— meaning formation of products is unfavorable.

    • This is because 3-phosphoglyceric acid is very low in free energy that glyceraldehyde-3-phosphate dehydrogenase could not phosphorylate it with just inorganic phosphate (it would need ATP, but the enzyme does not use ATP).

  • Thus, if 3-phosphoglyceric acid is formed, the desired product cannot be formed.

  • Thus, formation of 3-phosphoglyceric acid will not occur in this step. This is all theoretical. 

Describe what actually occurs during step 1 of phase 3 of glycolysis

1. The active site of glyceraldehyde-3-phosphate dehydrogenase has a cysteine residue. The SH of cysteine attacks the carbonyl carbon of glyceraldehyde-3-phosphate, forming a covalent bond. This forms a thioester intermediate between the enzyme and G3P/GAP. This oxidizes G3P/GAP because the bond to sulfur oxidizes the carbonyl carbon (its OD # goes up), and NAD+ is reduced to NADH. 

• Forming this thioester intermediate instead of 3-phosphoglyceric acid allows the product to actually form because the thioester intermediate is high enough in free energy that glyceraldehyde-3-phosphate dehydrogenase, with inorganic phosphate, can phosphorylate it. It is the same reaction just with a thioester intermediate instead of 3-phosphoglyceric acid. 

2. Glyceraldehyde-3-phosphate dehydrogenase uses a high-energy inorganic phosphate (which attacks the thioester intermediate) to phosphorylate the thioester intermediate. This forms 1,3-biphosphoglycerate. 

Describe what occurs during step 2 of phase 3 of glycolysis

Phosphoglycerate kinase removes the phosphate group from 1,3-biphosphoglycerate and adds it to ADP to yield ATP and 3-phosphoglycerate. 

• Note that this makes the product that we would have theoretically made in the previous step if it wasn’t so low in free energy 

  • Phosphoglycerate kinase is able to make this product because it can bind ATP and use its energy to form 1,3-biphosphoglycerate from such a low energy molecule (3-phosphoglycerate). 

• H+ is also required

• This reaction is reversible 

• The enzyme is named after the reverse reaction because 3-phosphoglycerate is more stable than 1,3-biphosphoglycerate 

• Because we go in with 2 GAPs, 2 ATP’s are formed. However, because we used 2 ATP’s in phase 1 with the kinases, the net gain of ATP is 0. 

Describe what occurs during step 3 of phase 3 of glycolysis

3-phosphoglycerate becomes 2-phosphoglycerate via phosphoglycerate mutase. 

• Phosphoglycerate mutase has a phosphohistidine in its active site. 

1. The phosphohistidine donates its phosphate group to carbon 2, which forms the intermediate 2,3-biphosphoglycerate (2,3-BPG). 

   • Recall that 2,3-BPG, which is produced as an intermediate in cells undergoing glycolysis, stabilizes the T-state of hemoglobin and causes it to release O2. This is good because cells undergoing glycolysis will eventually need O2 for the ETC chain so that they can get energy (ATP).

2. The phosphate on C3 is removed, which forms 2-phosphoglycerate. The phosphohistidine in phosphoglycerate mutases’s active site reforms. 

• Note that the phosphate group on C2 is not the same one that was on C3. It is from the phosphohistidine of the active site of phosphoglycerate mutase. 

• Note that this is NOT an isomerization reaction because the molecule is not converted from aldehyde to ketone or vice versa. 

Describe what occurs during steps 4 and 5 of phase 3 of glycolysis

Step 4:

2-phosphoglycerate loses H2O (via a dehydration reaction) with enolase and forms phosphoenolpyruvate. 

• Phosphoenolpyruvate has a C-C double bond with an O bound to one of them— recall that is what enols are 

• Phosphoenolpyruvate, like 1,3-biphosphoglycerate, is very likely to donate/lose its phosphate group. 

• Is reversible

Step 5:

Phosphoenolpyruvate, with pyruvate kinase and ADP, loses its phosphate group and becomes pyruvate. The phosphate group is added to ADP and produces ATP (substrate-level phosphorylation). 

• 1 ATP is produced for every phosphoenolpyruvate, so 2 ATP’s are produced per glucose, thus making our net gain of ATP 2. 

• This reaction is irreversible— pyruvate cannot be converted back to phosphopoenolpyruvate. 

• The enzyme is called pyruvate kinase because pyruvate is more stable than phosphoenolpyruvate. 

Describe the structure of pyruvate 

Is a 3-carbon molecule with 3 carbon functional groups— carboxylate, carbonyl, and methyl

List all of the irreversible reactions in glycolysis (in order)

1. Step 1 of phase 1— phosphorylation of glucose via hexokinase and 1 ATP. Forms glucose-6-phosphate. 

2. Step 3 of phase 1— phosphorylation of fructose-6-phosphate via phosphofructokinase and 1 ATP. Forms fructose-1,6-biphosphate. 

3. Step 5 of phase 3— dephosphorylation of phosphoenolpyruvate via pyruvate kinase to form pyruvate. Yields ATP because ADP is combined with the inorganic phosphate of phosphoenolpyruvate. 

Note that all of these reactions have a negative delta G— meaning they occur spontaneously (in the presence of ATP), release energy, and the formation of products is very favored. Most of the free energy decreases of glycolysis occur at these 3 steps, making them irreversible. In other words, these reactions are irreversible because the change in free energy is so large between reactants and products.

Interestingly, they are kinases.

What are the 3 fates of pyruvate? 

1. If O₂ is present, pyruvate will go into the citric acid cycle (continue through cellular respiration) and be converted to acetyl CoA and NAD+ will be converted to NADH via the pyruvate dehydrogenase complex (through a series of many oxidation reactions). This will produce a lot ATP. 

2. If O2 is not present, there are two fermentation options depending on the organism:

   1. Ethanol fermentation 

   2. Lactic acid fermentation 

Our main source of ATP comes from glycolysis if oxygen is low. Thus, the two pathways that occur in low oxygen conditions are all done to replenish the molecules required for glycolysis so that we can generate more energy— specifically, they replenish NAD+, which is a cofactor for certain enzymes in glycolysis (specifically glyceraldehyde-3-phosphate dehydrogenase). 

Describe what occurs during ethanol fermentation

• This only occurs in yeast and other microorganisms

• Has two steps:

  • Pyruvate is decarboxylated by pyruvate decarboxylase— so it loses its carboxyl group. This releases CO₂ and forms acetaldehyde.

    • This step is irreversible.

  • Acetaldehyde is reduced to ethanol by alcohol dehydrogenase. This produces ethanol and NAD+ because the reduction of acetaldehyde is coupled with the oxidation of NADH (which was formed in glycolysis), thereby releasing NAD+. 

Thus, NAD+ is regenerated so that it can return to phase 3 of glycolysis and be reduced to NADH by GAP dehydrogenase so that glyceraldehyde-3-phosphate can be oxidized to form the thioester intermediate. This will allow glycolysis to occur again and produce 2 ATP during these low O₂ conditions.

Describe what occurs during lactic acid fermentation

This occurs in animals (including humans) and some microorganisms. In humans, it occurs during intense physical activity in which O₂ is very limited in skeletal muscle cells.

Has 1 step:

• Pyruvate is reduced (loses a bond to oxygen) by lactate dehydrogenase and forms lactate/lactic acid.

• This oxidizes NADH (which comes from glycolysis), creating NAD+

• This is reversible 

Thus, pyruvate is reduced to lactic acid in low O2 conditions in skeletal muscle cells so that NAD+ can be regenerated so that it can return to phase 3 of glycolysis and be coupled with the oxidation of GAP and be reduced to NADH. This allows us to make 2 ATP from glycolysis under low oxygen conditions.