Enzyme catalysed reactions of glycolysis step by step (glycolysis part 2)

Enzyme catalysed reactions of glycolysis step by step

In the first half of glycolysis, energy in the form of two ATP molecules is required to transform glucose into two three-carbon molecules.

 Step 1: Phosphorylation of glucose (First priming reaction)

A glucose molecule is energized and activated for subsequent reactions by the addition of a high-energy phosphate from ATP, forming glucose-6-phosphate.

Enzyme catalysing the reaction: Hexokinase (Requires Mg2+ cofactor)

Type of the reaction: Phosphoryl transfer

Phosphoryl group donor: ATP

Nature of the reaction: Irreversible under intracellular conditions

Phosphorylation site on glucose: Hydroxyl group on C-6 of glucose

(Kinases are enzymes that catalyze the transfer of the terminal phosphoryl group from ATP to an acceptor nucleophile. The acceptor in the case of hexokinase is a hexose, normally D-glucose, although hexokinase also catalyzes the phosphorylation of other common hexoses, such as D-fructose and D-mannose)

Why the Hexokinase require Mg2+ ion as cofactor?

  • Because the true substrate of the enzyme is not ATP4- but the MgATP2-
  • Mg2+ shields the negative charges of the phosphoryl groups in ATP, making the terminal phosphorus atom an easier target for nucleophilic attack by an OH of glucose.

This step is notable for two reasons:

(1) Glucose 6-phosphate cannot diffuse through the hydrophobic interior of the plasma membrane, because of its negative charges. The cell lacks transporters for G6P. So it can no longer leave the cell.

(2) The addition of the phosphoryl group begins to destabilize glucose, thus facilitating its further metabolism.

Additional point to remember: This reaction consumes ATP, but it acts to keep the glucose concentration low, promoting continuous transport of glucose into the cell through the plasma membrane transporters.

Step 2: Conversion of Glucose 6-Phosphate to Fructose 6-Phosphate

Isomerization of glucose 6-phosphate (an aldose) to fructose 6-phosphate (a ketose)

Enzyme catalysing the reaction: Phosphohexose isomerase (phosphoglucose isomerase)

Type of the reaction: Isomerization

Nature of the reaction: Reversible under normal cell conditions

However, it is often driven forward because of a low concentration of F6P, which is constantly consumed during the next step of glycolysis. Under conditions of high F6P concentration, this reaction readily runs in reverse.

(This isomerization has a critical role in the overall chemistry of the glycolytic pathway, as the rearrangement of the carbonyl and hydroxyl groups at C-1 and C-2 is a necessary prelude to the next two steps. The phosphorylation that occurs in the next reaction (step 3 ) requires that the group at C-1 first be converted from a carbonyl to an alcohol, and in the subsequent reaction (step 4 ) cleavage of the bond between C-3 and C-4 requires a carbonyl group at C-2).

Step 3: Phosphorylation of Fructose 6-Phosphate to Fructose 1, 6- bisphosphate (Second priming reaction)

PFK -1 catalyzes the transfer of a phosphoryl group from ATP to fructose 6-phosphate to yield fructose 1, 6-bisphosphate

Enzyme catalysing the reaction: Phosphofructokinase-1 (PFK-1)

Type of reaction: Phosphoryl transfer

Phosphoryl group donor: ATP

Nature of the reaction: Irreversible under cellular conditions

It is the first “committed” step in the glycolytic pathway; glucose 6-phosphate and fructose 6-phosphate have other possible fates, but fructose 1, 6-bisphosphate is targeted for glycolysis.

(Phosphofructokinase -1 is a regulatory enzyme. It is the major point of regulation in glycolysis).

Additional information:

The prefix bis- in bisphosphate means that two separate monophosphate groups are present, whereas the prefix di- in diphosphate (as in adenosine diphosphate) means that two phosphate groups are present and are connected by an anhydride bond.

Step 4: Cleavage of Fructose 1, 6-Bisphosphate

  • This is the “lysis” step that gives the pathway its name.
  • The modified sugar fructose-1, 6-bisphosphate is unstable, which allow it to split in half and form two phosphate-bearing three-carbon sugars (triose phosphates)
  • Fructose 1, 6-bisphosphate is cleaved to yield two different triose phosphates:
  1. Glyceraldehyde 3 phosphate (an aldose)
  2. Dihydroxy acetone phosphate (a ketose)

Enzyme catalysing the reaction: Fructose 1, 6-bisphosphate aldolase (simply aldolase)

Type of reaction: Aldol cleavage or aldol condensation

Nature of the reaction: Reversible

(Aldolase: This enzyme derives its name from the nature of the reverse reaction, an aldol condensation).

Step 5: Interconversion of the Triose Phosphates

Type of reaction: Isomerization

  • Only one of the two triose phosphates formed by aldolase (glyceraldehyde 3-phosphate) can be directly degraded in the subsequent steps of glycolysis.
  • The other product, dihydroxy acetone phosphate, is rapidly and reversibly converted to glyceraldehyde 3-phosphate by triose phosphate isomerase.

(Isomerase catalyzes the reversible conversion between the two three-carbon sugars. This reaction never reaches equilibrium in the cell because the next enzyme in glycolysis uses only glyceraldehyde-3-phosphate as its substrate (and not dihydroxy acetone phosphate). This pulls the equilibrium in the direction of glyceraldehyde-3-phosphate, which is removed as fast as it forms. Thus, the net result of steps 4 and 5 is cleavage of a six-carbon sugar into two molecules of glyceraldehyde-3-phosphate; each will progress through the remaining steps of glycolysis).

Here ends the first phase of glycolysis.

 To summarize: In the preparatory phase of glycolysis the energy of  2 ATP is invested, raising the free-energy content of the intermediates, and the carbon chains of all the metabolized hexoses are converted into a common product, glyceraldehyde 3-phosphate.

The payoff phase of glycolysis (Phase of energy generation)

The energy gain comes in the payoff phase of glycolysis.

Step 6: Oxidation of Glyceraldehyde 3-Phosphate to 1, 3-Bisphosphoglycerate

Enzyme catalysing the reaction: Glyceraldehyde 3- phosphate dehydrogenase

Type of reaction: Phosphorylation coupled to oxidation

Nature of the reaction: Reversible

The reaction catalyzed by glyceraldehyde 3- phosphate dehydrogenase is really the sum of two processes:

1) The oxidation of the aldehyde to a carboxylic acid by NAD+

2) The joining of the carboxylic acid and orthophosphate to form the acyl-phosphate product

 Explanation in detail

First reaction:  The sugar is oxidized by the transfer of electrons and H+ to NAD+, forming NADH (a redox reaction).

  • The acceptor of hydrogen in the glyceraldehyde 3- phosphate dehydrogenase reaction is NAD+
  • H+ ion from the aldehyde group of glyceraldehyde 3-phosphate reduces the coenzyme NAD+ to NADH.
  • The other hydrogen atom of the substrate molecule is released to the solution as H+

This reaction is very exergonic, and the enzyme uses the released energy to attach a phosphate group to the oxidized substrate, making a product of very high potential energy. The source of the phosphates is the pool of inorganic phosphate ions that are always present in the cytosol.

  • 1, 3-Bisphosphoglycerate is an acyl phosphate (carboxylic acid anhydride with phosphoric acid). Such compounds have a high phosphoryl-transfer potential.
  • One of its phosphoryl groups is transferred to ADP in the next step in glycolysis.

Step 7: Formation of ATP from 1, 3-Bisphosphoglycerate

 Type of reaction: Phosphoryl transfer

Nature of the reaction: Reversible

Enzyme catalysing the reaction: Phosphoglycerate kinase

 Reaction: Enzyme catalyzes the transfer of the phosphoryl group from the acyl phosphate of 1, 3-

bisphosphoglycerate to ADP. ATP and 3-phosphoglycerate are the products.

Steps 6 and 7 of glycolysis together constitute an energy-coupling process in which 1, 3-bisphosphoglycerate is the common intermediate.

The outcomes of the reactions catalyzed by glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase are:

  1. Glyceraldehyde 3-phosphate (an aldehyde) is oxidized to 3-phosphoglycerate (a carboxylic acid).
  2. NAD+ is reduced to NADH.
  3. ATP is formed from Pi and ADP at the expense of carbon oxidation energy.

The formation of ATP by phosphoryl group transfer from an organic substrate such as 1, 3-bisphosphoglycerate is referred to as a substrate-level phosphorylation.

(Reason: The phosphate donor (1, 3-BPG) is a substrate with high phosphoryl-transfer potential).

Point to remember: Because of the actions of aldolase and triose phosphate isomerase, two molecules of glyceraldehyde 3- phosphate were formed from single glucose and hence two molecules of ATP were generated. These ATP molecules make up for the two molecules of ATP consumed in the first stage of glycolysis.

Additional information

(Substrate-level phosphorylations involve soluble enzymes and chemical intermediates (1, 3-bisphosphoglycerate in this case). Respiration-linked phosphorylations, on the other hand, involve membrane-bound enzymes and transmembrane gradients of protons).

Step 8: Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate

 Type of reaction: Phosphoryl shift

Enzyme catalysing the reaction: Phosphoglycerate mutase

Nature of the reaction: Reversible

 Reaction: Intramolecular shift of the phosphoryl group between C-2 and C-3 of glycerate.

This step prepares the substrate for the next reaction. Mg2+ cofactor is essential for this reaction

Step 9: Dehydration of 2-Phosphoglycerate to Phosphoenolpyruvate

Type of the reaction: Dehydration

Nature of the reaction: Reversible

Enzyme catalysing the reaction: Enolase

 Reaction: Removal of a molecule of water from 2-phosphoglycerate to yield phosphoenolpyruvate (PEP)

 This enzyme causes a double bond to form in the substrate. The electrons of the substrate are rearranged in such a way that the remaining phosphate bond becomes very unstable, preparing the substrate for the next reaction. An enol phosphate has a high phosphoryl-transfer potential.

 Why does phosphoenolpyruvate have such a high phosphoryl-transfer potential?

The phosphoryl group traps the molecule in its unstable enol form. When the phosphoryl group has been donated to ATP, the enol undergoes a conversion into the more stable ketone namely, pyruvate.

Step 10: Transfer of the Phosphoryl Group from Phosphoenolpyruvate to ADP

Type of reaction: Phosphoryl transfer

Nature of the reaction: Irreversible

Enzyme catalysing the reaction: Pyruvate kinase

 Reaction: Transfer of the phosphoryl group from phosphoenolpyruvate to ADP, which requires K+ and either Mg2+ or Mn2+.

The products of the reaction is 2 ATP molecule/glucose and 2 pyruvate/glucose

(Pyruvate is the ionized form of pyruvic acid)

ATP generation occurs via substrate level phosphorylation

Since the molecules of ATP used in forming fructose 1,6-bisphosphate have already been regenerated, the two molecules of ATP generated from phosphoenolpyruvate are “profit.”

Overview of glycolytic pathway

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