Cytosol [Ref^. Harper 26/e, p 136]

• Conversion of 2 molecules of glyceraldehyde 3 phosphate to 1,3 bisphoglycerate release 2 molecules of NADH which yield 6 ATP.
• Conversion of 2 molecules of 1,3 BPG to 3 phosphoglycerate yield 2 ATP.
• Conversion of 2 molecules of phosphoenol pyruvate to pyruvate yield 2 ATP.

• Conversion of glucose to glucose 6 phosphate 
• Conversion of fructose 6 phosphate to fructose 6 bis phosphate
• Total ATP formed during glycolysis  : 10
• ATP utilised  during glycolysis          :   2
• Net ATP formed during glycolysis    :   8

• Aerobic glycolysis: is a process of splitting of glucose into two molecules of pyruvate with the synthesis of ATP.
• Anaerobic Glycolysis: glycolysis is the only process in which ATP is generated anaerobically. This is of importance because RBC which does not have a mitochondria is wholly dependant on the anaerobic energy production. The byproduct of anaerobic glycolysis is lactate.

• Rate limiting step: Phosphofructokinase the commited step of glycolysis.
• Irreversibles steps:

ATP formation in glycolysis:

Ref: Bender D.A., Mayes P.A. (2011). Chapter 18. Glycolysis & the Oxidation of Pyruvate. In D.A. Bender, K.M. Botham, P.A. Weil, P.J. Kennelly, R.K. Murray, V.W. Rodwell (Eds), Harper’s Illustrated Biochemistry, 29e.

Although there are two oxidation-reduction steps is the anaerobic glycolysis, and the energy for the synthesis of two ATP molecules is released, there is no net change in the oxidation state of carbon. The first oxidative reaction is catalyzed by glyceraldehyde-3-phosphate dehydrogenase to yield NADH. Later, NADH is spent for the reduction of pyruvate to the lactate which is catalyzed by lactate dehydrogenase. The ratio between C, H, and O atoms is the same for both glucose, C6H12O6 and lactic acid C3H6O3.

The most potent positive allosteric activator of phosphofructokinase-1 and inhibitor of fructose 1,6-bisphosphatase in the liver is fructose 2,6-bisphosphate.

• It relieves inhibition of phosphofructokinase-1 by ATP and increases the affinity for fructose 6-phosphate. 
• It inhibits fructose 1,6-bisphosphatase by increasing the Km for fructose 1,6-bisphosphate. 
• Its concentration is under both substrate (allosteric) and hormonal control (covalent modification).

Glycolysis, the major pathway for glucose metabolism, occurs in the cytosol of all cells. Glycolysis is both the principal route for glucose metabolism and also the main pathway for the metabolism of fructose, galactose, and other dietary carbohydrates. 

Hexokinase catalyzes the conversion of glucose to glucose-6-phosphate in the energy-requiring first step of glycolysis. ATP is also required in the conversion of fructose-6-phosphate to fructose 1,6-bisphosphate by PFK. ATP is generated in the conversion of 1,3-bisphosphoglycerate to 3-phosphoglycerate by phosphoglycerate kinase and in the conversion of phosphoenolpyruvate to pyruvate by PK. Both phosphoenolpyruvate carboxykinase and pyruvate carboxylase are energy-requiring reactions except that these occur in the gluconeogenesis pathway.

A, B, C i.e. Enolase, Phospho-glyceromutase, Glyceraldehyde-3-phosphate-dehydrogenase

1. C i.e. Conversion of glucose to 3 C units

• Enzymes of glycolysis – a process which converts 6 carbon glucose to 3 carbon unit pyruvate & lactateQ – are present in cytoplasm. Whereas complete oxidation of glucose (to CO) & H₂0) requires mitochondrial enzymes (of TCA cycle).
• Out of total 9 enzymes used in glycolysis

–   3 enzymes – hexokinase, phosphofructokinase (PFK-1) and Pyruvate kinase are used in irreversible steps.

–   6 enzymes – phosphohexose isomerase, aldolase, glyceraldehydes 3 phosphate dehydrogenase, 1, 3- biphospho glycerate kinase, phosphoglyceromutase and enolase are used in reversible stepsQ.

–   2 enzymes used in energy utilizing steps are – hexokinase (using 1 ATP) and phosphofructokinase PFK-1 (using 1 ATP). This energy consumption is for 1 molecule of glucose.

–  3 enzymes used in energy producing steps are – glyceraldehydes 3phosphate dehydrogenase (producing 2 NADH = 5 ATP), 1, 3 – biphospho glycerate kinase (producing 2 ATP), and pyruvate kinase (producing 2 ATP). This energy production is for 1 molecule of glucose or 2 molecules of glyceraldehyde 3-P.

C i.e. Glucose 6 PO4

Glucose 6 phosphate is an important compound that joins several metabolic pathways viz. glycolysis, glycogenolysis, glycogenesis, gluconeogenesis and pentose phosphate pathwayQ

All of the above

D i.e. Phosphofructokinase

Ans:A i.e. Phosphofructokinase.

Rate-Limiting Enzymes 

• Rate-limiting enzyme of Glycolysis :Phosphofructokinase-1 (PFK-1)
• Rate-limiting enzyme of Gluconeogenesis :Fructose-1,6,biphosphatase
• Rate-limiting enzyme of TCA cycle :Isocitrate dehydrogenase
• Rate-limiting enzyme of Glycogen Synthesis :Glycogen synthase
• Rate-limiting enzyme of Glycogenolysis :Glycogen phophorylase (phophorylase breaks phosphate bond, which means activated glycogen releases a lot of energy)
• Rate-limiting enzyme of HMP Shunt :Glucose-6-Phosphate dehydrogenase (bad to lose this in RBCs)
• Rate-limiting enzyme of de novo pyrimidine synthesis :Carbamoyl phosphate synthase II (CPS I is involved in urea cycle)
• Rate-limiting enzyme of de novo purine synthesis :Glutamine-PRPP amidotransferase
• Rate-limiting enzyme of Urea cycle :Carbamoyl phosphate synthetase I (CPS II is involved in pyrimidine synthesis)
• Rate-limiting enzyme of fatty acid synthesis :Acetyl-CoA carboxylase (ACC)
• Rate-limiting enzyme of fatty acid oxidation :Carnitine acyltransferase I
• Rate-limiting enzyme of Ketogenesis :HMG-CoA synthase
• Rate-limiting enzyme of Cholesterol synthesis :HMG-CoA reductase

A i.e. Phosphofructokinase

A i.e. Cytosol

The activation as well as the quantities of certain key enzymes of glycolysis, namely glucokinase (NOT hexokinase), phosphofructokinase and pyruvate kinase are increased by insulin.

Glycolysis/Embden-Meyerhof pathway is the sequence of reactions that converts glucose into pyruvate with the concomitant production of a relatively small amount of adenosine triphosphate (ATP)

It is the initial process of most carbohydrate catabolism, and it serves three principal functions:

• Generation of high-energy molecules (ATP and NADH) as cellular energy sources as part of aerobic respiration and anaerobic respiration.
• Production of pyruvate for the citric acid cycle as part of aerobic respiration
• Production of a variety of six- and three-carbon intermediate compounds, which may be removed at various steps in the process for other cellular purposes

In eukaryotes and prokaryotes, glycolysis takes place within the cytosol of the cell.

Ans.’ B’ Lactate

When a muscle contracts under anaerobic conditions, glycogen disappears and lactate appears. When oxygen is admitted, aerobic recovery takes place and lactate is no longer produced. If muscle contraction occurs under aerobic conditions, lactate does not accumulate and pyruvate is the end product of glycolysis. Pyruvate is oxidized further to CO₂ and water.

Seven of the reactions of glycolysis are reversible and are used in the synthesis of glucose by gluconeogenesis.

Thus, seven enzymes are common to both glycolysis and gluconeogenesis :

1. Phosphohexose isomerase;
2. Aldolase;
3. Phosphotriose isomerase,
4. Glyceraldehyde 3-phosphate dehydrogenase;
5. Phosphoglycerate kinase;
6. Phosphoglycerate mutase;
7. Enolase.

Three reactions of glycolysis are irreversible which are circumvented in gluconeogenesis by four reactions. So, enzymes at these steps are different in glycolysis and gluconeogenesis.

Ans. ‘D’ 0

Net number of ATPs produced from 1 mol of Glucose by

•  Anaerobic Glycolysis- 2 ATPs
•  Aerobic Glycolysis –  7 ATPs
•  Aerobic oxidation-   32 ATPs
•  Rapoport-LueberingCycle-  Zero

Ans. is ‘b’ i.e., Phosphoglycorate kinase

Glycolysis is regulated at 3 steps which are irreversible.

These reactions are catalyzed by following key enzymes :‑

1) Hexokinase and glucokinase

2) Phosphofructokinase – I

3) Pyruvate kinase.

Ans. is ‘a’ i.e., 4 ATP, 2 NADH

Enegetics of glvcolysis

During glycolysis 2 ATP are utilized and 4 ATP are produced at substrate level. 2 reducing equalents NADH’ are produced and reoxidized by electron transport chain, to generata 5 ATP molecules (2.5 ATP per NADH’ molecule). Thus total 9 ATP molecules are produced and 2 are utilized, i.e., There is net gain of 7 ATP molecules in aerobic glycolysis.

In anaerobic conditions, the reoxidation of NADH by electron transport chain is prevented and NADH gets reoxidized by conversion of pyruvate to lactate by lactate dehydrogenase. Thus, in anaerobic glycolysis only 4 ATP are produced at substrate level. Therefore, there is net gain of 2 ATP molecules in anaerobic glycolysis.

Note : – Previous calculations were made assuming that NADH produces 3 ATPs and FADH2 generates 2 ATPs. This will amount to a net generation of 8ATPs per glucose molecule during glycolysis. Recent experiments show that these old values are overestimates and NADH produces 2.5 ATPs and FADH2 produces 1.5 ATPs. Thus, net generation is only 7ATPs during glycolysis.

Ans ‘C’ 4

During anaerobic (oxygen deficient) condition, when one molecule of glucose is converted to 2 molecules of lactate, there is a net yield of 2 molecules of ATP.

Ans.B

Regulation of glycolysis

• Glycolysis is regulated at 3 irreversible steps. These reactions are catalyzed by following key enzymes : (1) Hexokinase and glucokinase, (2) Phosphofructokinase I, and (3) Pyruvate kinase.

Hexokinase and glucokinase

These enzymes catalyze the first step of glycolysis, i.e., Glucose —> Glucose-6-phosphate. Glucokinase is found in the liver, whereas hexokinase is found in all tissues. The kinetic properties of these two are different.

Hexokinase has low Km, i.e., high affinity for glucose, low Vmax, and is subjected to feedback inhibition by the reaction product, glucose-6-phosphate. Hexokinase is found in most of the tissue except the liver and comes into play when blood glucose is low. It is not affected by feeding or insulin. Hexokinase is not specific for glucose metabolism, it is also involved in the metabolism of fructose and galactose.

Glucokinase, on the other hand, is specific for glucose. It has high Km (i.e., low affinity for glucose), high Vmax and unlike hexokinase, it is not inhibited by glucose-6-phosphate. As it has a low affinity for glucose (high km), it comes into play only when intracellular glucose concentration is high. It is induced by feeding and insulin. Glucagon inhibits glucokinase.

The function of hexokinase is to provide glucose-6-phosphate at a constant rate, according to the needs of cells, i.e., the function of hexokinase is to provide constant glucose utilization by all tissues of the body even when blood sugar is low. The function of glucokinase in the liver is to remove glucose from blood after a meal, providing glucose-6­phosphate over the requirement for glycolysis so that it can be used for glycogen synthesis and lipogenesis.

Phosphofructokinase I

• Phosphofructokinase I is the major regulatory enzyme of glycolysis. It catalyzes the 3rd reaction of glycolysis, i.e., fructose-6-P Fructose 1,6 bis-P. This reaction is irreversible and is the “rate-limiting step” for glycolysis. It is also the “committed step”, meaning that once fructose 1,6 bisphosphate is formed it must go for the glycolytic pathway only. So, the most important control point for glycolysis is through the regulation of phosphofructokinase I.
• Phosphofructokinase-I is allosterically activated by Fructose-6-phosphate, fructose 2,6-bisphosphate, AMP, ADP, K^+, and phosphate. It is allosterically inhibited by ATP, citrate, Ca+2, Mg+2, and low pH. Phosphofructokinase is an inducible enzyme that increases its synthesis in response to insulin and decreases in response to glucagon.
• Fructose 2,6-bisphosphate (F-2,6-BP) is the most important allosteric modulator (activator) of phosphofructokinase-I. Fructose 2,6-bisphosphate is synthesized as a side product of glycolysis. A bifunctional enzyme named PFK-2/Fructose 2,6 bisphosphatase is responsible for regulating the level of fructose 2,6 bisphosphate in the liver. Phosphofructokinase-2 (PFK-2) activity of this bifunctional enzyme is responsible for the synthesis of F-2,6-BP from fructose-6-phosphate and fructose 2,6 bisphosphatase activity is responsible for the hydrolysis of F-2,6-BP back to fructose-6-phosphate.

Ans. is ‘d’ i.e., Phosphotriose isomerase 

Enzyme in gluconeogenesis

• Seven of the reactions of glycolysis are reversible and are used in the synthesis of glucose by gluconeogenesis. Thus, seven enzymes are common to both glycolysis and gluconeogenesis: (i) Phosphohexose isomerase; (ii) Aldolase; (iii) Phosphotriose isomerase; (iv) Glyceraldehyde 3-phosphate dehydrogenase; (v) Phosphoglycerate kinase; (vi) Phosphoglycerate mutase; (vii) Enolase.
• Three of the reactions of glycolysis are irreversible and must be circumvented by four special reactions which are unique to gluconeogenesis and catalyzed by : (i) Pyruvate carboxylase, (ii) PEP carboxykinase, (iii) Fructose-1, 6- bisphosphatase, (iv) Glucose-6-phosphatase.

Ans. is ‘d’ i.e., Phosphoglycerate kinase 

• Seven of the reactions of glycolysis are reversible and are used in the synthesis of glucose by gluconeogenesis. Thus, seven enzymes are common to both glycolysis and gluconeogenesis: (i) Phosphohexose isomerase; (ii) Aldolase; (iii) Phosphotriose isomerase, (iv) Glyceraldehyde 3-phosphate dehydrogenase; (v) Phosphoglycerate kinase; (vi) Phosphoglycerate mutase; (vii) Enolase.
• Three reactions of glycolysis are irreversible which are circumvented in gluconeogenesis by four reactions. So, enzymes at these steps are different in glycolysis and gluconeogenesis.

Reactions                                       Enzyme in glycolysis        Enzyme in gluconeogenesis

Glucose – Glucose-6-P                    Hexokinase/glucokinase   Glucose-6-phosphatase

Fructose-6-P – Fructose- I ,6-BP     Phosphofructokinase         Fructose-1-6-bisphosphatase

Phosphoenolpyruvate – Pyruvate    Pyruvate kinase                Pyruvate carboxylase PEP carboxykinase

Ans. is ‘c’ i.e., Two carbon end product is formed 

Important facts about glycolysis

• An important biochemical significance is the ability of glycolysis to provide ATP in the absence of oxygen (anerobic glycolysis) and allows tissues to survive anoxic episodes.
• It occurs in cytosol
• 3 Carbon atoms end product (pyruvate or lactate) is produced.
• Irreversible steps are catalyzed by : – Glucokinase/Hexokinase, phosphofructohnase-I, and pyruvate kinase. 
• Reversible steps are catalyzed by : – Phosphohexose isomerase, aldolase, phosphotriose isomerase, glyceraldehyde 3-phosphate dehydrogenase, Phosphoglycerate kinase, Phosphoglycerate mutase, Enolase.
• Energy (ATP) using steps are catalyzee by : – Hexokinase/glucokinase, phosphofurctokinase.
• Energy (ATP) production at substrate level are catalyzed by : Phosphoglycerate kinase, Pyruvate kinase. 
• Reducing equivalent (NADH) production is catalyzed by : Glyceraldehyde 3-phosphate dehydrogenase.
• Cancer cells derive nutrition from glycolysis as they have lack of 02 supply because of lack of capillary network. Glycolysis (anaerobic glycolysis) is the only metabolic pathway in the body which can provide energy by glucose metabolism in anerobic conditions.

Ans. is ‘c’ i.e., Brain 

There are two types of glycolysis : –

1.  Aerobic glycolysis : – It occurs when oxygen is plentiful and the final product is pyruvate, i.e., final step is catalyzed by pyruvate kinase (see the cycle above). Which is later converted to acetyl CoA by oxidative decarboxylation. There is net gain of 7 ATPs. Acetyl CoA enters TCA cycle.
2. Anaerobic glycolysis : – It occurs in the absence of oxygen. The pyruvate is fermented (reduced) to lactate in single stage. The reoxidation of NADH (formed in the glyceraldehyde-3-phosphate dehydrogenase step) by respiratory chain is prevented as same NADH is utilized at lactate dehydrogenase step. So, there is no net production of NADH. Thus, there is net gain of 2 ATP only. Unlike pyruvate which is converted to acetyl CoA to enter into krebs cycle, lactate cannot be further utilized by further metabolic pathways. Thus, lactate can be regareded as dead end in glycolysis. Anaerobic glycolysis occurs in exercising skeletal muscle, RBCs, lens, some region of retina, renal medulla, testis and leucocytes.

Ans. is ‘c’ i.e., Malate shuttle 

• Most of the NADH and FADH2, entering the mitochondrial electron transport chain arise from citric acid cycle and 13-oxidation of fatty acids, located in the mitochondria itself.
• However, NADH is also produced in the cytosol during glycolysis.
• To get oxidized, NADH has to be transported into the mitochondria as respiratory chain (ETC) is located inside the mitochondria.
• Since, the inner mitochondrial membrane is not permeable to cytoplasmic NADH, there are special shuttle systems which carry reducing equivalents from cytosolic NADH (rather than NADH itself) into the mitochondria by an indirect route.
• Two such shuttle systems that can lead to transport of reducing equivalent from the cytoplasm into mitochondria are : –

1. Malate shuttle (malate-aspartate shuttle system).
2. Glycerophosphate shuttle.

Ans. is ‘b’ i.e., Pasteur effect 

Pasteur effect

• It has been observed that under anaerobic condition a tissue or microorganism utilizes more glucose than it does under aerobic conditions.
• It reflects inhibition of glycolysis by oxygen and is called pasteure effect.
• The Pasteur effect is due to inhibition of the enzyme phosphofructokinase because of inhibitory effect caused by citrate and ATP, the compounds produced in presence of oxygen due to operation of TCA cycle. Crabtree effect
• This is opposite of Pasteur effect, which represents decreased respiration of cellular systems caused by high concentration of glucose.
• When oxygen supply is kept constant and glucose concentration is increased, the oxygen consumption by cells falls, i.e., relative anaerobiosis is produced when glucose concentration is increased in constant supply of oxygen.
• It is seen in cells that have a high rate of aerobic glycolysis.
• In such cells the glycolytic sequence consumes much of the available Pi and NAD⁺, which limits their availability for oxidative phosphorylation.
• As a result, rate of oxidative phosphorylation decreases, and oxygen consumption also shows a corresponding fall.

Regulation of Carbohydrate Metabolism-

Ans. ‘B’ Hexokinase

Glucose enters glycolysis by phosphorylation to glucose-6-phosphate, catalyzed by hexokinase, using ATP as the phosphate donor.

Ans- ‘D’ Phosphofructokinase.

Glucose-6-phosphate is the junction of several metabolic pathways: glycolysis, gluconeogenesis, the pentose phosphate pathway, glycogenesis, and glycogenolysis.

In glycolysis, it is converted to fructose6-phosphate by phosphohexose isomerase, which involves an aldose– ketose isomerization. This reaction is followed by another phosphorylation catalyzed by the enzyme phosphofructokinase1 forming fructose 1,6-bisphosphate which is the committed step of glycolysis.

Ans- ‘A’ Phosphofructokinase

Phosphofructokinase-1 is the pace-setting enzyme of glycolysis.

It is inhibited by ATP and citrate, uses fructose 6-phosphate as substrate, and catalyzes a reaction that is far from equilibrium. The reaction is activated by fructose 2,6-bisphosphate.

The rate of fructose metabolism is more rapid than that of glucose because the trioses formed from fructose 1-phosphate bypass phosphofructokinase-1—the major rate-limiting step in glycolysis.

– In glycoslysis 4 ATPs are produced in which 2 ATPs are utilized so a net gain of 2 ATPs

2 NADH molecules are produced.

Ans-‘C’ Lactate

Aerobic tissues metabolize pyruvate to acetyl-CoA, which can enter the citric acid cycle for complete oxidation to CO2 and H2O. Glycolysis can also occur anaerobically (in the absence of oxygen) when the end product is lactate.