Biochemistry Chapter 16

Once lactate and pyruvate are produced they diffuse to the blood through carriers. Once in the blood what are the two fates?

1) Cardiac muscle and slow twitch cells contain carrier permeable to lactate and pyruvate, which their high oxygen allows lactate to be converted into pyruvate and processed by the citric cycle and oxydative phosphorylation. 2) Excess lactate enters the liver and is converted to pyruvate then into glucose through gluconeogenesis, restoring the glucose necessary for active cells. called the cori cycle.

Why is the closing of the cleft from hexokinase so important?

1) Favors the reaction between the hydroxyl group and the phosphoryl group of ATP because it makes glucose more nonpolar. 2) Keeps water away from the active site. This prevents water from attacking the ATP in the binding site of the CH2OH from the glucose. NOTE: this makes sense because water cleaves ATP, so any water found in the active site would cleave ATP.

What are the three outcomes from which ATP is created using the enzymes 3-phosphate dehydrogenase and phosphoglycerate kinase:

1) Glyceraldehyde is oxidized to 3-phosphateglycerate. 2) NAD+ is reduced to NADH 3) ATP is formed from Pi and ADP. NOTE: since this occurs twice, two molecules of ATP wre created. Also in this oxydation, energy is temporally trapped as 1,3 BPG.

What inhibits/enhances activity of phosphofructokinase?

1) High levels of ATP allosterically inhibit it. By binding to a site that is not the active site, lowers the affinity of the enzyme to substrate. 2) High levels of AMP increases the activity of this enzyme. NOTE: When there is high energy (ATP) the hyperbolic curve changes to a sigmoidal. NOTE the ATP/AMP ratio.

What are two features of TPI are very important:

1) Is a great example of a kinetically perfect enzyme, with a Kcat/Km ration of 2X10^8 m^-1s^-1 2) Supressess the decomposition of enediol intermediate into methyl glyoxal and orthophosphate, which is very toxic, that attack proteins and DNA. The enzyme prevents the exit of enediol by using a loop that acts as a lid over the top of the active site and opens when isomerization is complete.

How is galactose converted into glucose?

1) Phosphorylaton of galactose into galatose 1-phosphate by galactokinase 2) Galactose 1-P acquire a uridyl group from UDP glucose, which is an activated intermediate in the synthesis of carbs . Enzyme Galactose 1-phosphtate uridyl transferase 3) UDP galactose 4-epimerase converts UDP galactose into UDP glucose. 4) glucose 1-phosphate is ismerized to 6 phosphate glucose by Phosphoglucomutase.

Describe the mechanism of the conversion of GAP into 1,3 BPG.

1) The aldehyde reacts with the sulfhydryl group of cysteine 149 on the enzyme to form a hemithioacetal. 2) A hydride ion is transfered to NAD+ thats tightly bound to the enzyme. This reaction is favored because His 176 deprotonates the hemithioacetal. 3) NADH leaves and thioester intemediate is formed and another NAD+ arrives. The + on NAD polyrizes the intermediate to facilitate attack by orthophosphate. 4) Pi attacks the thioester and frees the Cys residue, forming 1,3 BPG

why does the phosphoenolpyrubate (PEP) have such a high phosphory-transfer potential?

1) The phosphoryl group traps the molecule in its unstable enol form. When the phosphoryl group has been donated to ATP, the molecule is free and converts to a more stable ketone-pyrubate.

why does glucose gets phosphorylated so many times?

1) if cleavage takes place in the aldose glucose, Two metabolic pathways are need to process the two carbon and four carbon fragmented products. 2) phosphorylation of fructose 6-phosphate to fructose 1,6-biphosphate prevents it from going back to glucose 6-phosphate. Therefore, the process yields two Phosphorylated interconvertible three-carbon fragments.

How are the concentration of ATP, ADP and AMP in the cell?

1) is constant of the short term 2) [ATP] > [ADP] and [ADP] > [AMP] NOTE: small concentration changes of ATP lead to large changes in the concentration of AMP and ADP which increases the range of sensitivity of PFK.

After ATP is converted for the first time in glycolysis. What happens next:

3-phophoglycerate is converted into pyruvate, and another ATP is formed.

Describe how TIP catalyses the formation of GAP from DHAP?

A glutamate residue plays a role of an acid-base catalyst but is not basic enough to pull a proton from C-1. Histidine assits by donating a proton to stabilized the negative charge that develops on the C-2 carbonyl group.

In glycolysis, What follow the isomerization step? And what is the end product of this? What enzyme is involved?

A second phosphorylation happens. Fructose 6-Phosphate gets phosphorylated At the expense of ATP to fructose 1,6-biphosphate. Phosphofructokinase (PFK), an allosteric enzyme.

What factors stimulate PFK and what stimulates fructose 1,6-bisphosphatase?

AMP stimulates PFK and inhibits fructose 1,6-bisphosphatase, thus glycolysis is turned on gluconeogenesis down. HIgh levels of ATP and citrate inhibiting PFK and stimulate fructose 1,6-bisphosphatase. NOTE: high levels of ATP and Citrate indicate high energy charge.

What molecule is used to power the addition of CO2 to pyruvate? What happens when Co2 is removed?

ATP. But remember that GTP is the phosphate group donor. Removal of the CO2 group to poer the formation of phosphoenolpyruvate in the decarboxylation. NOTE: IN general, decarobxylation drive reaction that are highly endergonic.

What is the function of Acetyl CoA?

Acetyl CoA provides the entry point to oxidative pathway to extract more energy from glucose after fermentation, which is formed inside the migochondria by oxydative decarboxylation of pyruvate.

Define Fermentation:

An ATP generating process in which organic compounds act both as the donor and acceptor of electrons.

Briefly define the molecule of 1,3 BPG:

An energy rich molecule with a greater Phosphoryl transfer potential than that of ATP. Thus, 1,3 BPG can be used to power the synthesis of ATP From ADP.

How are PFK2 and FBPase2 regulated in the liver?

Are reciprocally controlled by the phosphorlation of a single serine residue. LOW glucose levels: rise of glucagon in the blood trigger cyclic AMP signal cascade leading to the phosphorylation of this protein by protein kinase A activating FBPase2 and stimulating gluconeogenesis. NOTE: glucagon also inactivates pyruvate kinase. HIGH glucose: insuline activates a protein phosphatase, which removes a phosphoryl group from the enzyme activating PFK2 and accelerating glycolysis by F-2,6-BP.

What is the function of GLUT1 and GLUT3?

Basal glucose uptake. KM values of 1, allowing essential and continual uptake of glucose into cells.

Why do we say that there is not net oxydation-reduction in the conversion of pyruvate to ethanol?

Because NADH is generated by the oxydation of glyceraldehyde 3-phosphate and is consumed in the reduction of acetaldehyde to ethanol.

Why is AMP used as a regulator indicator rather from ADP?

Because energy can be extracted from two ADP molecules with the enzyme adenylate kinase, which gives ATP and AMP ( low energy state)

Why is PFK preferred to be regulated that hexokinase?

Because glucose6-Phosphate is a intermediate that can be converted to different things. Where as the phosphorylation of fructose 6-Phosphate is irreversible.

why is the generation of NAD+ from the reduction of pyrubate to lactate or ethanol beneficial?

Because it sustains the continued process of glycolysis under anaerobic conditions.

Why is the carboxylation and a decarvoxylation required to form phosphoenolpyruvate from pyruvate?

Because the addition of a phosphoryl group to pyruvate is highly unfavorable. (Delta G'=+31). Therefore the addition of CO2 and deCO2 steps result in a more favorable delta G' =+.8kj mol.

What how does Pyruvate kinase gets inhibited/enhanced?

By ATP allosterically. Slow the enzyme down when energy charge is high. Enhanced: When glycolysis increases, Fructose 1,6-biphosphate activates the kinase.

What is the importance of phosphoglycerate kinase?

Catalyses the transfer of phosphoryl group from the acyl phosphate of 1,3 BPG to ADP. ATP and 3-Phosphoglycerate are the products. NOTE: the formation of ATP in this manner is referred to as substrate level phosphorylation because 1,3 BPG has a HPTP.

What inhibits PFK in the liver? Why not ATP or pH?

Citrate, an early intermediate in the citric cycle. High concentrations in the cytoplams means that biosynthetic precursor are abundant, and no more need to degrade glucose. Inhibits allosterically like ATP. liver does not suffer from lactic acid or ATP needs.

What is an allosteric regulator for pyruvate carboxylase?

CoA, which enhances the formation of carboxybiotin.

What is the following step after the phosphorylation of fructose 6-phosphate to fructose 1,6-biphosphate by the enzyme phosphofructokinase? Which enzyme is involved?

Fructose 1,6 biphosphate is cleaved into GAP and DHAP which completes stage 1, which consist of three carbons. Aldolase, A lyase enzyme.

What enhances the activity of PFK in the liver?

Fructose 2,6-bisphosphate, which produced when there is a high concetration of fructose 6-phosphate, which is produced when there is a high glucose concentration. Binding of F-2,6-BP increases the affinity of PFKto its substrate and diminishes the inhibitory effect of ATP.

GLUT5

Fructose transporter in small intestines.

What happens to fructose in other tissues like adipose tissue?

Gets phosphorylated to fructose 6-phosphate by hexokinase.

After glucose 6-phosphate is produced, what is the next step to produce glucose in the glyconeogenic pathway?

Glucose 6-phosphate is process to glucose by glucose 6-phosphatase, which is present only in tissues that maintail metabolic homeostasis such as the liver and kidneys.

What is the only fuel that the brain use and the only fuel for red blood cells?

Glucose.

Define glycolysis:

Glycolysis is the sequence of reactions that metabolizes one molecule of glucose into two molecules of pyruvate with the net production of two molecules of ATP. NOTE: the three fates of glucose

briefly describe glycolysis:

Glycolysis takes place in the cytoplasm in eukaryotes, and comprises two stages: 1) trapping and preparation phase: No ATP can be generated. The strategy is to trap glucose in the cell and form a compound that can be cleaved into phosphorylated three-carbon units. 2) ATP is generated when the three carbon fragments are oxydized to pyruvate.

What is the enzyme that transfers a phosphoryl groups from ATP to the hydroxyl group on a carbon 6 of glucose? What is the purpose of this?

Hexokinase. transfers a phosphoryl group from ATP to a variety of six-carbon sugar, hexoses (glucose and mannose) forms glucose 6-phosphate. This compound cannot pass through the membrane because of the negative charges and is not substrate for glucose transporters. Also facilitates its metabolism to three carbon molecules.

Hexokinase and glucokinase in the liver are inhibited by?

Hexokinase: same as in muscle. Glucokinase: liver specific proteins only when glucose levels are low. Glucokinase provides glucose 6-phosphate for synthesis of glycogen and fatty acids and displays sigmoidal kinetics eventhough it functions as a monomer. its affinity for glucose is low, therefore its only active when glucose is high.

In what part of the cell does the hydrolysis of glucose 6-phosphate takes place?

In the lumen of the ER, Glucose 6-phosphatase is located in the membrane. Then, Glucose and Pi are transported to the cytoplasm by a pair of transporters.

after the function of phosphoglycerate mutase enzyme, what is the following reaction in the second stage of the glycolytic cycle? What enzyme is involve in this reaction?

In the second stage of the glycolytic cycle, dehydration of the 2-phosphoglycerate, creating PEP. Enzyme: Enolase. NOTE: an enol phosphate has a high Phosphoryl transfer potential than an ester of an ordinary alcohol such 2-phosphglycerate.

What is the first step of the glycolytic pathway second phase? This reaction can be viewed as the sum of two process, what are these two processes?

Is the conversion of GAP to 1,3 BPG. catalyzed by the enzyme glyceraldehyde 3-phosphate dehydrogenase. 1) The oxydation of the aldehyde to a COOH by NAD+ and 2) jointing the COOH and orthophosphate to form the Acyl phosphate product (1,3 BPG).

What happens after oxolacetate is formed?

Is transported to the cytoplasms by reducing it to malate by malate dehydrogenase, which is transported across the mitchondrial membrane and reoxidized to oxaloacetate by cytoplasmic NAD+-linked makate dehydrogenase. This creates NADH which is used in gluconeogenesis. Then, oxyloacetate is simultaneously decarboxylated and phosphorylated by phosphophenolpyrubate carboxykinase. GTP is phosphoryl donor.

What is the following step after hexokinase activity? How?

Isomerization of glucose 6-phosphate to fructose 6-phosphate. This converts the glucose 6 phosphate aldose into a fructose 6-Phosphate ketose

Why does the thioester intermediate efficiently reacts with orthophosphate to form 1,3 BPG,

It is a high energy compound, and preserves all the energy released by the oxydation reduction. NOTE: on the picture you can see how the same reaction compare with and without intermediate.

how hexokinase activity increased and reduced?

Its inhibited by its own product glucose 6-Phosphate by negative feedback. This signals that the cell does not need glucose for energy or for glycogen synthesis.

What is the funstion of GLUT4?

KM of 5mM. Transports glucose into muscle and fat cells. GLUT4 increases rapidly in the presence of insulin. endurance training also increases it.

How does hexokinase catalyzes the transfer of a phosphoryl group?

Like other kinases, this enzyme requires Mg2+ for activity. It consists of two lobes that close when glucose binds. This enclosing glucose except for the hydroxyl group of the C-6, which is the phosphoryl acceptor. NOTE: This is an example of induced fit.

So how does Fructose gets metabolized? 3 steps

Metabolized in the liver using fructose 1-phosphate pathway. 1) Phosphorylation of fructose to fructose 1-phosphate. Enzyme fructokinase 2) Fructose 1-phosphate is split into glyceraldehyde and dihydroxyacetone phosphate. enzyme; fructose 1-phosphate aldolase 3)Phosphorylation of glyceraldehyde to glyceraldehyde 3-phosphate by triose kinase.

Is gluconeogenesis a reversible process? What about equilibrium and energy change.

NO! This means is not the reversal of glycolysis. Equilibrium lies to the formation of pyruvate. Mos of the decrease in free energy in glycolysis takes place in three irreversible steps: see picture.

Are there any catbolic pathways for metabolizing fructose?

NO!!!!

Over consumption of fructose has been linked to fatty liver, insulin insensitivity, and obesity. What factors contribute to this?

Note fructokinase and triose kinase bypass the most important regulatory step in glycolysis the phosphokinase catalyzed reactions, Fructose derived glyceraldehyde 3-phosphate and dihydroxyacetone are processed by glycolysis to pyrubate and to Acetyl CoA in a unregulated fashion. Excess acetyl CoA to fatty acids which can be transported to adipose tissue, resulting in obesity. The activity of the enzymes can deplete ATP. The liver can also accumulate the fatty acids from Acetyl CoA.

Describe how ethanol is produced from Pyruvate?

Occurs in yeast and bacteria. 1) decarboxylation of pyruvate by pyruvate decarboxylase. This enzyme requires pyrophosphate, a coenzyme from vitamin thiamine. 2) Reduction of acetaldehyde to ethanol by NADH catalyzed by alcohol dehydrogenase. Generates NAD+ NOTE: this reaction is an example of alcoholic fermentation.

What is the most important control site of the glycolytic pathway?

Phosphofructokinase.

What enzyme is responsible for the conversion of glucose 6-phosphate to fructose 6-phosphate? why is this so complicated?

Phosphoglucose isomerase. This takes several steps because of the presence of both sugar in their cyclic forms. Therefore the enzyme must first open the glucose ring, perform isomerization, then form the five member ring of fructose.

How is the interconversion of pyruvate and phosphoenolpyruvate reciprocally regulated in the liver?

Pyruvate kinase is inhibited by ATP and alanine indicating high energy charge. ADP inhibits pyrubate carboxylase and phosphoenolpyruvate carboxylase anc activated by Acetyl CoA.

What enzyme catalyzes the transfer of the phosphoryl group from PEP to ADP? And how much ATP is created when glucose is converted into pyruvate?

Pyruvate kinase. Two molecules of ATP and two molecules of pyruvate are created.

What can you say about the conversion of glucose from the glycolytic and gluconeogenic pathway in terms of energy?

See picture and not the differences. The extra four high phosphoryl transfer potential are needed to turn an unfavorable process to an unfavorable one. A clear example of a coupling reaction. NOTE: glycolysis and gluconeogenesis are both exergonic.

What is the function of the enzyme phosphoglycerate mutase?

TThis enzyme causes a rearrangement of 3-phosphoglycerate into 2-phosphoglycerate. Requires large amounts of 2,3 BPG to maintain an active site histidine in a phosphorylated form. A phosphoryl is transfere 3-phosphoglycerate to reform 2,3-BPG. This enzyme functions as phosphatase because it removes the phosphate group from the C3- phosphate group from the 2,3 BPG to be converted into 2-phosphoglycerate.

What is feedfroward stimulation?

The acceleration of glycolysis due to the abundance of glucose.

What is the first step of gluconeogenesis?

The carboxylation of pyruvate at the expense of ATP by Pyruvate carboxylase in the mitochondria.

What is the definition of gluconeogenesis? and why is this important?

The conversion of glucose from pyruvate, which is converted from noncarbohydrate precursors, such as lactate, amino acids, and glycerol. or enter the pathway at later intermediates such as oxaloacetate and dihydroacetone phosphate. This helps maintain the glucose levels in the blood so that the brain and muscle extract sufficient to meet their metabolic demands.

What are the three reaction of pyruvate that are of importance?

The conversion of pyruvate into ethanol, lactate, CO2, and Acetyl CoA. NOTE: the first two reactions are fermentations that take place in the absence of oxygen.

what is galactosemia? And what is a common symptom?

The disruption of galactose metabolism by the deficientcy of 1 phosphate uridyl transferase. Common symptom is cataract formation, because aldose reductase cause the accumulating galactose to be reduced to galactitol, which accumulates in the lens.

What is the primary control of muscular glycolysis?

The energy charge of the cell, in other words the ratio of ATP to AMP.

In the formation of 1,3 BPG two reactions are required, What is the advantage of coupling them together? and how are they coupled?

The first reaction is very favorable (-50 kJ) whereas the second is not (+50KJ). Coupling enables the first reaction to drive the second one. They are coupled by a thioester intermediate formed as a result of the aldehyde oxydation.

What is so unique about fatty acids in gluconeogenesis?

The hydrolysis of triglycerols yield glycerol and fatty acids. Then glycerol can enter either the gluconeogenic or glycolytic pathway at dihydroacetone phosphate remember: Glycerol is a precursor of glucose. NOTE: animals cannot convert fatty acids to glucose.

What are the major sites for gluconeogenesis?

The liver and small region of the kidneys.

What happens when lactate starts to build up in muscles (fast twitch muscles)?

The pH of the muscles lower from 7 to 6.3. The drop of Ph inhibits phosphofructokinase.

Define gluconeogenesis:

The production of glucose from pyruvate, lactate, or amino acids.

What is lactic fermentation?

The reduction of pyruvate to lactate is catalyzed by lactase dehydrogenase

What is the importance of a substrate cycle?

They amplify metabolic signals. Which is made possible by the rapid hydrolysis of ATP. Glycolytic pathways increases by 1000-fold at the initiation of exercise.

What is so striking about PfK2 and FBPase2?

They are within the same polypeptide chain. Contains an N-terminal regulatory domain, Kinase domain and phosphatase domain

What is so common about the three dehydrogenases (glyceraldehyde 3-phosphate dehydrogenase, lactate and alcohol dehydrogenase?

They have different 3D structure but similar NAD+ binding domains. Which is made of four alpha helices and a sheet of six parallel beta strands.

How is the concentration of fructose 2,6-bisphosphate controlled to rise and fall with blood glucose levels ?

Two enzymes 1) PFK2 = produces F-2,6-BP 2) Fructose bisphosphatase 2 (FBPase2)=hydrolyses F-2,6-BP to F-6-P

How is pyruvate kinase inhibited in the liver?

Two forms of this enzyme: L in liver and M in muscle. In addition to allosteric inhibition the liver enzyme is inhibited by alanine and reversible phosphorylation. When glucose is low, the glucagon triggered cyclic AMP cascade leads to phosphorylation of this enzyme, decreasing its activity. This prevents the consumption of glucose by the liver when needed by the brain and muscle.

In the liver, How are the rates of glycolysis and gluconeogenesis regulated to maintain blood-glucose-levels?

Using the molecule of fructose 2,6-bisphosphata which strongly stimulates PFK and inhibits fructose 1,6-bisphosphatase.

How is hexokinase and phoshphofructokinse related in terms of regulation?

When PFK is inactive fructose 6-phosphate rise which causes the concetration of glucose 6-phosphate to rise due to equilibrium. NOTE: inhibition of PFK leads to hexokinase inhibition.

What is the warburg effect or aerobic glycolysis? Why is this so interesting

When reapidly growing tumors process glucose to lactate with or without oxygen. Acidification of tumor helps its invasion and inhibit the immune system.

What does the hypoxia that some tumor experience enhances?

activates transcription factor HIF-1, which increase the expression of GLUT1 and 3 and glycolytic enzymes until blood vessels can grow.

Why is UDP glucose not consumed in the conversion of galactose into glucose?

because is regenerated from UDP-galactose. The conversion of UDP glucose to galactose is essentiial for the synthesis of galatocyl residues in complex polysaccharides and in glycoproteins .

Fermentation Yields only a fraction of energy from glucose, meaning that is not as efficient, but why is so extensively used?

because it does not require oxygen.

What is the main cause of lactose intolerance?

deficiency of enzyme lactase, which normally reduces from 5 to 10% of the level at birth. Microorganism in the colon use lactose as energy and ferment it to lactic acid while generating methane, and H2. The lactate produced and undigested lactose are osmotically active and draws water into the intestine resulting in diarrhea.

What type of intermediate is involved in the catalyzation of DHAP into GAP?

enediol intermediate. NOTE: TIP catalyses the transfer of an H from C1 to C2, an intramolecular-oxidation reduction.

How does alanine become a precursor of glucose like lactate?

generated in the muscle when amino acids are used as fuels. N's from these aminos are transferred to pyruvate to form alanine. Takes place in the liver This also maintains Nitrogen balance.

Other than regulating FBPase2 and PFK2 what other things does glucagon and insulin regulate?

insulin: expressed after eating. Stimulates the expression of PFK, pyruvate kinase, and the bifunctional enzyme. glucagon: rises during fasting. inhibits expression of glycolytic enzymes and stimulates PEP carboxykinase and Fructose 1,6- bisphoshatease.

Why does pyruvate carboxylase requires biotin?

is a covalently attached prosthetic group that serves as an activated carrier of CO2. Carboxylate group is attached to biotin to the E-amino group of a lysine residue. Understand that carboxylation of pyruvate takes place in three steps: see picture.

Explain the structure of pyruvate carboxylase?

is a tetramer of four identical subunits with four domains. 1) Biotin carboxylase domain = catalyzes the formation of carboxyphosphate and the subsequent attachment of CO2 to the second domain BCCP 2) Once bound to CO2, BCCP biotin leaves the carboxylase active site and swigns to the active site of PC which transfers CO2 to pyruvate to form oxaloacetate. 3) The PT facilitates the formation of the tetramer.

What happens after phosphoenolpyruvate is formed in the gluconeogenic pathway?

is digested by enzymes of the glycolysis pathway until the next irreversible step, which is the hydrolysis of fructose 1,6-bisphosphasphate to fuctose 6-phosphate by the enzyme fructose 1,6bisphosphatase (allosteric enzyme which participates in the regulation of gluconeogenesis.

What is the purpose of TPI enzyme?

isomerizes the conversion of DHAP to GAP because DHAP cannot be used as GAP to get ATP. Instead they are both isomer. NOTE: At equilibrium DHAP predominates, but the reaction goes to the right because GAP is being removed due to glycolysis.

pH also affects phosphofructokinase activity, why is this beneficial?

pH falls when muscles are working anaerobically, the inhibitory effect protects the muscle from damage.

What is the function of GLUT2

present in liver and pancreatic B cells. High KM values for glucose allowing fast uptake of glucose. Allows the pancreas to sense the amount of glucose and adjust the level of insulin. Ensures that glucose enters the liver only in times of plenty.

What is a general feature of kinases like hexokinase?

substrate induced cleft closing.

In metabolic pathways, what type of enzymes are potential sites of control? Name the ones in glycolysis:

those that catalyze irreversible reactions. Glycolysis: Hexokinase, Phosphofructokinase, and pyrubate kinase. NOTE: this enzymes become more active or less in response to covalent modification or allosteric effectors.