Chapter 16 Glycolysis and Glucongeogenesis

Step 7 of glycolysis

1,3 - BPG is catalyzed by phosphoglycerate kinase to become 3 - phosphoglycerate. In this process ATP is also generated. Formation of ATP in this manner is referred to as substrate level phosphorylation because the phosphate donor, 1,3 - BPG, is a substrate with high phosphoryl - transfer potential.

The process of glycolysis results in the formation of two high-energy phosphate bonds (in the form of ATP). How many high-energy phosphate bonds are consumed during the process of gluconeogenesis using pyruvate as a starting material?

6 (four ATP and two GTP are needed to produce glucose from pyruvate)

Glucose labeled with carbon 14 at C-1 is incubated with the glycolytic enzyme and necessary cofactors. A) what is the distribution of Carbon 14 in the pyruvate that is formed? B) if the specific activity of the glucose substrate is 10 mCi mmol, what is the specific activity of the pyruvate that is formed?

A) the label is the methyl carbon atom of pyruvate. B) 5mCi. The specific activity is halved because the number of moles of product (pyruvate) is twice that of the labeled substrate (glucose).

Pyruvate

Can be further processed anaerobically to lactate (lactic acid formation) or ethanol (alcoholic fermentation)

Pathways that fructose enters the process of glycolysis

Can take one of two pathways. The first step is the phosphorylation of fructose to fructose 1 - phosphate by Fructokinase. Fructose 1 - phosphate is split into glyceraldehyde and dihydroxyacetone. (catalyzed by fructose 1 - phosphate aldolase). Glyceraldehyde is phosphorylated to glyceraldehyde 3 - phosphate (by triose kinase.)

Why does phosphoenolpyruvate have such a high phosphoryl transfer potential?

The phosphoryl group traps the molecule in its unstable enol form.

Explain step 2 of glycolysis

The second step is the isomerization of glucose 6 - phosphate to fructose 6 - phosphate. This involves a conversion of an aldose into a ketose. The enzyme (phosphoglucose isomerize) must first open the six - membered ring of glucose 6 - phosphate, catalyze isomerization, and then promote the formation of the five membered ring of fructose 6 - phosphate.

What is the strategy of the initial steps of glycolysis?

To trap glucose in the cell and form a compound that can be readily cleaved into phosphorylated 3 - carbon units

Mechanism: Triose Phosphate Isomerase (DHAP to G3P)

Triosephosphate isomerase (TPI) catalyzes the transfer of a hydrogen atom from carbon 1 to carbon 2. (an intramolecular oxidation.) This isomerization of a ketose proceeds through an enediol intermediate. Gluamate 165 plays the role of a general acid - base catalyst. It abstracts a proton (H+) from carbon 1 and then donates it to carbon 2. Histidine 95 assists catalysis by donating a proton to stabilize the negative charge that develops on the C - 2 carbonyl group.

Step 9 of glycolysis

2 - phosphoglycerate is catalyzed by enolase and becomes phosphenolpyruvate.

Step 8 of glycolysis

3 - phosphoglycerate undergoes rearragment to become 2 - phosphoglycerate. This reaction is catalyzed by phosphoglycerate mutase. Phosphoglycerate mutase requires catalytic amounts of 2,3 - bisphosphoglycerate to maintain an active site histidine residue in a phosphorylated form. Phosphroyl group is transferred to reform 2,3 - BPG and the mutase functions as a phosphatase to form 2 - phosphoglycerate.

NAD+ is utilized in glycolysis during the transformation of Glyceraldehyde 3 - phosphate to 1,3 - Bisphoshoglycerate. NAD+ is reduced to NADH but for glycolysis to continue there must be more NAD+. How is NAD+ regenerated?

A fermenting organism can regenerate NAD+ at a later stage, when making ethanol. After pyruvate become Acetaldehyde. Acetaldehyde with the help of alcohol dehydrogenase becomes Ethanol and in the process regnerates NAD+ NAD+ can also be regenerated in the formation of Lactate. The reduction of pyruvate by NADH to form lactate is catalyzed by lactate dehydrogenase which in the process regenerates NAD+.

In the reaction Fructose 1,6 - Bisphosphate an Aldolase is utilized to yield Glyceraldehyde 3 - phosphate and DHAP. What would have occured if glucose were to be cleaved by Aldolase?

A two carbon and four carbon fragment would have resulted. Two different metabolic pathways, one to process the two - carbon fragment and one for the four - carbon fragment, would have required to extract energy.

mutase

An enzyme that catalyzes the intramolecular shift of a chemical group.

Well into exercise, the ratio of ATP/AMP in muscle cells decreases. What effect does this ratio reduction have on the activity of phosphofructokinase and pyruvate kinase?

Both enzymes decrease in activity due to lack of allosteric binding of ATP

A suspension of yeast cells is being grown under anaerobic conditions such that glucose is degraded to ethanol and carbon dioxide. If one wishes to follow this process by monitoring the release of 14CO2, at which positions in the glucose molecule would the 14C label need to be incorporated?

Carbons 3 and 4 will end up as the carboxyl group of pyruvate during glycolysis. this is the source of the released carbon dioxide.

Amylase

Cleaves the alpha - 1,4 bonds of starch and glycogen, but not the alpha - 1,6 bonds. The products are maltose and maltotrisose. The material not digestible because of the alpha - 1,6 bonds is called the limit dextrin.

Active Site of alcohol dehydrogenase

Contains a zinc ion that is coordinated to the sulfur atoms of two cysteine residues and a nitrogen atom. This zinc ion polarizes the carbonyl group of the substrate to favor the transfer of a hydride from NADH.

Pyruvate kinase (muscle)

Controls the outflow from glycolysis. ATP allosterically inhibits pyruvate kinase to slow glycolysis when the energy charge is high. Alanine also allosterically inhibits pyruvate kinase, to signal that building blocks are abundant.

Step 5 of glycolysis

Dihydroxyacetone (DHAP) also must be converted into G3P. This is accomplished by Triosephosphate isomerase. This leaves in combination of the previous step two molecules of G3P

Lactate

Formed from pyruvate is a process called lactic acid fermentation. The reduction of pyruvate by NADPH to form lactate is catalyzed by lactate dehydrogenase. The rengeration of NAD+ in the reduction of pyruvate to lactate or ethanol sustains the continued process of glycolysis under anaerboic conditions.

Step 4 of glycolysis

Fructose 1,6 - Bisphosphate is converted to Dihydroxyacetone (DHAP) and Glyceraldehyde 3 - Phosphate (G3P). The enzyme aldose is the catalysis for this step.

Step 3 of glycolysis

Fructose 6 - phosphate is phosphorylated at the expense of ATP to fructose 1,6 - bisphosphate. This is catalyzed by (phosphofructokinase)

Hexokinase (liver)

Glucokinase phosphorylates glucose only when glucose is abundant. The role glucokinase is to provide glucose 6 - phosphate for the synthesis of glycogen and for the formation of fatty acids.

What is the net reaction from glucose to pryuvate?

Glucose + 2pi + 2ADP + 2NAD+ --> 2 pyruvate + 2ATP + 2NADH + (2 H+) + 2H2O

Overall reaction of Lactate

Glucose + 2pi + 2ADP --> 2 lactate + 2ATP + 2H2O

Net result of alchoholic fermentation (creating ethanol)

Glucose + 2pi + 2DP + (2H+) --> 2 ethanol + 2CO2 + 2ATP + 2 H2O

Which steps of glycolysis are essentially thermodynamically irreversible?

Glucose to Glucose 6 - phosphate and Fructose 6 - phosphate to Fructose 1,6 - Bisphosphate. These reactions are essentially irreversible because they utilize ATP and are the energy investment phases of glycolysis.

Phosphofructokinase (muscle)

High levels of ATP allosterically inhibit the enzyme. The binding of ATP lowers the enzyme's affinity for fructose 6 - phosphate. AMP reverses the inhibitor action of ATP. A decrease in pH also inhibits phosphofructokinase activity by augmenting the inhibitory effect of ATP.

Explain the first step of glycolysis

In the first step of glycolysis Glucose is phosphoryalted by ATP to from glucose 6 - phosphate. Glucose 6 - phosphate cannot pass through the membrane because it is not a substrate for the glucose transporters. The addition of the phosphoryl group acts to destabilize glucose. This transfer of a phosphate from ATP to carbon 6 is catalyzed by hexokinase.

phosphofructokinase (liver)

In the liver, phosphofructokinase is inhibited by citrate. A high level of citrate in the cytoplasm means that precursors are abundant, and there is no need to degrade additonal glucose. Citrate inhibits phosphofructokinase by enhancing the inhibitory effects of ATP.

Fermentation

Is an ATP generating process in which organic compounds act as both donor and as acceptors of electrons.

Hexokinase

Kinases are enzymes that transfer a phosphoryl group from ATP to an acceptor. Hexokinase requires Mg2+ for activity. The structure consists of two lobes, which move toward each other when glucose is bound. The cleft between the lobes closes, and the bound glucose becomes surrounded by protein, except for the hydroxyl group of carbon 6, which will accept the phosphoryl group from ATP.

The processes of gluconeogenesis and glycolysis are said to be reciprocally regulated. This means that

Molecules that inhibit (or activate) one process often have the opposite effects on the other.

When glyceraldehyde 3-phosphate oxidatively converted to 1,3-bisphosphglycerate in the presence of phosphate and glyceraldehyde 3-phosphate dehydrogenase, what is reduced?

NaD+, is the electron sink for glycolysis. When an intermediate is oxidized, NAD+ reduced to NADH.

Avidin, a 70 - Kd protein in egg white, has very high affinity for biotin. In fact, it is a highly specific inhibitor of biotin enzymes. Which of the following conversions would be blocked by the addition of avidin to a cell homogenate? (a) glucose --> Pyruvate (B) Pyruvate --> Glucose (C) Oxaloacetate --> glucose (D) Malate --> Oxaloacetate (E) Pyruvate --> Oxaloacetate (F) G3P --> Fructose 1,6 - Bisphosphate

Only reactions B and E would be blocked. Biotic is an important vitamin cofactor for gluconeogenesis. It supplies the carbon dioxide necessary for the carboxylation of pyruvate to form oxaloacetate. Thus option E will be blocked. The formation of glucose from pyruvate requires the conversion of pyruvate to oxaloacetate. So B would also be blocked

Pathway Galactose enters the process of glycolysis

Phosphorylation of galactose to galactose 1 - phosphate by galactokinase. Galactose 1 - phosphate then acquires a uridyl group from uridine diphosphate glucose (UDP - glucose). The products of this reaction, which is catalyzed by galactose 1 - phosphate uridyl transferase, are UDP - galactose and glucose 1 - phosphate. UDP - galactose is then epimerized to glucose.

Glucokinase (pancreas)

Present in B cells of the pancreas, where the increased formation of glucose 6 - phosphate by glucokinase when blood - glucose levels are elevated leads to the secretion of the hormone insulin.

How is phosphoenolpyruvate formed in gluconeogenesis?

Pryruvate is converted into oxaloacetate by the enzyme pyruvate carboxylase. Pyruvate carboxylase uses biotin, ATP, and activating acetyl COA to form oxaloacetate. This converts to malate and moves back to the cytoplasm, where it is re - oxidized to oxaloacetate. Phosphoenolpryuate carboxykinase converts oxaloacetate into phosphoenolpyruvate. The carboxyl that was added to pyruvate is now gone.

How does pyruvate become oxaloacetate?

Pryuvate to oxaloacetate would be the first reaction of gluconeogenesis which is the conversion from pyruvate to glucose. This first process is achieved by the enzyme pyruvate carboxylase. Pyruvate carboxylase uses biotin to carry CO2 and place it on the pyruvate to make oxaloacetate.

Gluconeogenesis

Pyruvate is converted into glucose. Several reactions must differ from glycolysis because the equilbirum of glycolysis lies on the side of pyruvate formation. Gluconeogenesis bypasses virtually irreversible reactions of glycolysis.

Besides Triosephosphate isomerase (TPI) catalyzes the reaction between DHAP to G3P what else does it do?

TPI also suppresses an undesired side reaction, the decomposition of the enediol intermediate into methyl glyoxal. TPI must prevent the enediol from leaving the enzyme. The intermediate is trapped in the active site by the movement of a loop of 10 residues. This loop serves as a lid on the active site, shutting it when the enediol is present and reopening when isomerization is complete.

Describe the active site of glyceraldehyde 3 - phosphate dehydrogenase and explain the mechanism of the phosphorylation coupling to the oxidation of G3P by a thioester intermediate.

The active site of G3PD includes a reactive cysteine residue, as well as NAD+ and a crucial histidine. Mechanism: 1. The aldehyde substrate reacts with the sulfhydryl group of cysteine 149 on the enzyme to form a hemithioacetal. 2. Is the transfer of a hydride ion to a molecule of NAD+ that is tightly bound to the enzyme and is adjacent to the cysteine residue. (products NADH and thioester intermediate) 3. The NADH formed leaves the enzyme and is replaced by a second molecule of NAD+. (the positive charge on NAD+ polarizes the thioester intermediate to facilitate the attack by orthophosphate) 4. Orthophosphate attacks the thioester to form 1,3 - BPG and free the cystein reside

In eukaryotic cells where does glycolysis take place?

The cytoplasm

1,3 - Bisphosphoglycerate

The energy released during the oxidation of G3P to 3 - phosphoglycerate is temporarily trapped as 1,3 - BPG. This energy provides the transfer of a phosphate from 1,3 - BPG to ADP to ATP. (substrate level phosphorylation)

Citric acid / ETC

The entry point to this oxidative pathway is acetyl coenzyme A. Formed inside mitochondria by the oxidative decarboxylation of pyruvate.

What is significant of glucose induced structural changes?

The enviroment around the glucose becomes more nonpolar and the conformation changes enable the kinase to discriminate against H2O as a substrate.

Hexokinase (muscle)

The enzyme that catalyzes the first step of glycolysis, is inhibited by its product, glucose 6 - phosphate. A rise in glucose 6 - phosphate concentration is a means by which phosphofructokinase communicates with hexokinase. When phosphofructokinase is inactive, the concentration of fructose 6 - phosphate rises. The level of glucose 6 - phosphate rises in response as it is in equilibrium with fructose 6 - phosphate. Which means that the inhibition of phosphofructokinase leads to the inhibition of hexokinase.

Pyruvate carboxylase

The enzyme that catalyzes the reaction of pyruvate into oxaloacetate. This reaction takes place inside the mitochondria. The N - terminus of the enzyme forms an ATP - grasp domain. This is an ATP - activating domain found in many enzymes. The C - terminus constitute a biotin - binding domain. Biotin is a covalently attached prosthetic group, which serves as a carrier of activated CO2

Why is phosphofructokinase rather than hexokinase the pacemaker of glycolysis?

The first irreversible reaction unique to the glycolytic pathway, the committed step is the phosphorylation of fructose 6 - phosphate to fructose 1,6 - bisphosphate.

Explain the step of gluconeogensis : the conversion of pyruvate into phosphoenolpyruvate

The first step costs a molecule of ATP and is catalyzed by pyruvate carboxylase. This converts pyruvate into oxaloacetate. The second part costs a molecule of GTP and is catalyzed by phosphoenolpyruvate carboxylase. This coverts oxaloacetate into phosphoenolpyruvate.

Ethanol

The first step is the decarboxylation of pyruvate which is catalyzed by pyruvate decarboxylase, which require the coenzyme thiamine pyrophosphate. The second step is the reduction of acetaldehyde to ethanol by NADPH. This is catalyzed by alcohol dehydrogenase and regenerates NAD+.

The two processes from step 6 of glycolysis: G3P to 1,3 - bisphosphoglycerate must be coupled so that the favorable aldehyde oxidation can be used to drive the formation of the acyl phosphate. How are these reactions coupled?

The key is an intermediate, formed as a result of the aldehyde oxidation, that is linked to the enzyme by a thioester bond.

Glycolysis

The sequences of reactions that metabolizes one molecule on glucose to two molecules of pyruvate with the concomitant net production of two molecules of ATP

If cells synthesizing glucose from lactate are exposed to CO2 labeled with C14, what will be the distribution of label in the newly synthesized glucose?

There will be no labeled carbons. The CO2 added to pyruvate (formed from the lactate) to form oxaloacetate is lost with the conversion of oxaloacetate into phosphoenolpyruvate.

Step 6 of glycolysis

This reaction can be seen as a sum of two processes. The first part is the oxidation of the aldehyde of G3P to carboxylic acid by NAD+. The second the joining of the carboxylic acid and orthophosphate to from the acyl - phosphate product. This conversion from G3P to 1,3 - Bisphosphoglycerate is catalyzed by Glyceraldehyde 3 - phosphate dehydrogenase.

Why is AMP and not ADP the positive regulator of phosphofructokinase?

When ATP is being utilized, the enzyme adenylate kinase can form ATP from ADP. AMP becomes the signal for the low - energy state.

Although glucose is the major sugar degraded by the glycolytic pathway, other sugars such as galactose and fructose are also derived from food. These sugars

are metabolized by the glycolytic pathway by conversion to intermediates in the pathway.

The three major regulatory enzymes in the glycolytic pathway are

hexokinase, phosphofructokinase, and pyruvate kinase.

What product of contracting muscle tissue is used by the liver as starting material for gluconeogenesis?

lactate

Step 10 of glycolysis

phosphenolpyruvate is catalyzed by pyruvate kinase and become pyruvate. The dehydration of 2 - phosphoglycerate introudces a double bond, creating an enol. When the phosphoryl group has been donated to ATP, the enol undergoes a conversion into the more stable ketone (pyruvate).

The mechanism of glyceraldehyde-3-phosphate dehydrogenase does NOT involve

phosphorylation of the substrate using ATP

If an inhibitor of alcohol dehydrogenase is added to the above suspension, the cells rapidly die because

the NAD needed to keep glycolysis going is not being recycled so the entire process is shut down and no ATP is produced.