8 Steps of Citric acid Cycle (Krebs cycle) and Enzymes involved in each Step

The Krebs cycle, Citric acid cycle or TCA cycle is an eight step cyclic reactions in which acetyl CoA is oxidized producing CO2, reduced coenzymes (NADH + H+ and FADH2), and ATP.

Site of Reaction: Mitochondrial matrix in Eukaryotes

                              Cytoplasm in Prokaryotes

All enzymes are present in mitochondrial matrix except succinate dehydrogenase which is bound to inner mitochondrial membrane

Pyruvate formed in Glycolysis enters mitochondrion and is converted to acetyl CoA which enters Krebs cycle.

Step 1: Condensation reaction

Acetyl CoA (C2) is added to oxaloacetate (C4) to form citrate (C6).

Step1 Enzyme: Citrate synthase

Coenzyme A (CoA-SH) is removed in the process.

Step 2: Isomerization

Citrate (C6) is isomerized forming isocitrate (C6).

Step2 Enzyme: Aconitase

2 step process: First step involves dehydration of citrate to cis-aconitase (unstable product)

Second step involving rehydration of cis-aconitase into isocitrate.

Step 3: Dehydrogenation (1^st NADH synthesis) and decarboxylation (reduction in C atom: C6 to C5)

Isocitrate (C6) is converted into alphaketoglutarate (C5).

Step3 Enzyme: Isocitrate dehydrogenase

2 step process: First step isocitrate is dehydrogenated to oxalosuccinate (C6) (intermediate product) NAD+ is reduced, forming NADH + H+

Second step: Decarboxylation of oxalosuccinate(C6) to α-ketoglutarate (C5). Carbondioxide is removed.

Step 4: Oxidative decarboxylation (2^nd NADH synthesis)

(reduction in C atom: C5 to C4)

α-ketoglutarate (C5) is oxidatively decarboxylated to form succinyl-CoA (C4), high energy compound.

Step 4 Enzyme: α-ketoglutarate dehydrogenase enzyme complex

During this step coenzyme A is added, CO2 is removed, and NAD+ is reduced, forming NADH + H+

Step 5: Substrate level phosphorylation (GTP/ATP synthesis)

Succinyl CoA(C4) is converted to succinate (C4)

Step 5 Enzyme: succinyl-CoA synthase

Coenzyme A is released, Substrate level phosphorylation of guanosine diphosphate (GDP) to guanosine triphosphate (GTP). GTP is then hydrolyzed to form ATP

Step 6: Dehydrogenation (FADH2 synthesis)

Succinate(C4) is converted to fumarate (C4).

Step 6 Enzyme: succinate dehydrogenase in inner mitochondrial membrane

During this step, FAD is reduced forming FADH2.

Step 7: Hydration

Fumarate(C4) is converted to malate(C4) with the addition of water.

Step 7 Enzyme: Fumarase (Fumaric acid hydratase)

Step 8: Dehydrogenation (3^rd NADH synthesis)

Final step: Malate (C4) is converted to oxaloacetate(C4)

Step 8 Enzyme: Malate dehydrogenase

In the process, NAD+ is reduced to form NADH + H+.

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Glycolysis Poster with Steps and Enzymes

 

Simple 10 step video on Glycolysis

Glycolysis- Enzymes and Regulatory Steps

10 steps of Glycolysis with enzymes

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3 Regulatory Enzymes and rate limiting step of Glycolysis

 Glycolysis (Glyco=Glucose; lysis= splitting) is the oxidation of glucose (C 6) to 2 pyruvate (3 C) with the formation of ATP and NADH.

It is also called as the Embden-Meyerhof Pathway
Glycolysis is a universal pathway; present in all organisms:
from yeast to mammals.

It is a universal anaerobic process where oxygen is not required.

Lear more on 10 steps of Glycolysis

In metabolic pathways, enzymes catalyzing essentially irreversible reactions are potential sites of control. In glycolysis, the reactions catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase are virtually irreversible; hence, these are the regulatory enzymes in Glycolysis.

This is our 5 minute video Regulatory Enzymes and rate limiting step of Glycolysis

Regulatory Enzyme 1 : Hexokinase

Step 1: Phosphorylation of glucose to glucose-6 phosphate (Hexokinase)

This reaction requires energy and so it is coupled to the hydrolysis of ATP to ADP and Pi.
• Enzyme: hexokinase. It has a low Km for glucose; hexokinase phosphorylates glucose that enters the cell
• Irreversible step. So the phosphorylated glucose gets trapped inside thecell. Glucose transporters transport only free glucose

Hexokinase Activators:AMP/ADP (indicating low energy or ATP therefore activates hexokinase

Inhibitors: Glucose-6-phosphateInhibitors

If G6P accumulates in the cell, there is feedback inhibition of hexokinase till the G6P is consumed

Regulatory Enzyme 2 and Rate limiting step : Phosphofructokinase (PFK)

Step 3: Phosphorylation of fructose-6- bisphosphate.(PFK)

Phosphorylation of the hydroxyl group on C1  forming fructose-1,6- bisphosphate.

Enzyme: phosphofructokinase. This allosteric enzyme regulates the pace of glycolysis (rate limiting step).

The rate limiting step is the slowest (irreversible) step in a pathway, which determines how fast the whole pathway can be carried out.

ATP is used
Second irreversible reaction of the glycolytic pathway.

PFK Activators: AMP/ADP, Fructose-2,6-bisphosphate

Inhibitors: ATP, Citrate

Citrate inhibits PFK by enhancing the inhibitory effect of ATP.

Fructose 2,6-bisphosphate (PFK-2) activates PFK by increasing its affinity for fructose 6-phosphate and diminishing the inhibitory effect of ATP 

This reaction is unique to Glycolysis therefore rate limiting step

Regulatory Enzyme 3: pyruvate kinase.

Step 10: Enolphosphate is a high energy bond. It is hydrolyzed to form the enolic form of pyruvate with the synthesis of ATP. Irreversible step

Enzyme: pyruvate kinase.

Enol pyruvate quickly changes to a more stable keto pyruvate.

Pyruvte kinase Activators:AMP/ADP , Fructose-1,6-bisphosphate

Inhibitors: ATP, Acetyl CoA, Alanine

If fructose 1,6 bisphosphate is formed, it acts a allosteric feedforward activator and drives the pyruvate kinase reaction forward.

Alanine, an aminoacid derived from pyruvate, is a negative
effector of catabolism.

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What is feed forward activation of enzymes? With example form Glycolysis

Definition: In Feed-forward activation, a metabolite produced early in a pathway activates an enzyme that catalyze a reaction further down the pathway

Feed Forward activation in Glycolysis

Pyruvate kinase (Step 10 enzyme) is activated by Fructose-1,6-bisphosphate (3 rd step metabolite)

Fructose-1,6- bisphosphate is formed by the phosphorylation of fructose-6- bisphosphate by the enzyme Phosphofructokinase (PFK). This is the committed step or rate limiting step of Glycolysis. Excess Fructose-1,6- bisphosphate in the rate limiting step activates the formation of final product of the reaction; pyruvate by activating the enzyme in the final step; pyruvate kinase.

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10 Steps of Glycolysis, Enzymes involved and Regulatory Enzymes of Glycolysis

Glycolysis (Glyco=Glucose; lysis= splitting) is the oxidation of glucose (C 6) to 2 pyruvate (3 C) with the formation of ATP and NADH.

• It is also called as the Embden-Meyerhof Pathway
• Glycolysis is a universal pathway; present in all organisms:
• from yeast to mammals.
• It is a universal anaerobic process where oxygen is not required
• First phase of cellular reparation in aerobic organisms
• It occurs in the cytosol of cell cytoplasm in both eukaryotes and prokaryotes

In the presence of O2, pyruvate is further oxidized to CO2.
In the absence of O2, pyruvate can be fermented to lactate or ethanol.
Net Reaction:

Glucose + 2NAD+ + 2 Pi + 2 ADP = 2 pyruvate + 2 ATP + 2NADH + 2 H2O

Here is the video that explains 10 Steps of Glycolysis 

2 stages of Glycolysis

First phase: Preparatory Phase or investment phase Phosphorylation of Glucose and its conversion to Glyceraldehyde 3-phosphate. 2 ATP used in this pahse

Second phase: Payoff phase

Oxidative conversion of Glyceraldehyde 3-phosphate to pyruvic acid

(4 ATP and 2 NADH produced)

10 Steps of Glycolysis

Reaction 1: Phosphorylation of glucose to glucose-6 phosphate

This reaction requires energy and so it is coupled to the hydrolysis of ATP to ADP and Pi.

Enzyme: hexokinase (regulatory step). It has a low Km for glucose; hexokinase phosphorylates glucose that enters the cell

Irreversible step. So the phosphorylated glucose gets trapped inside thecell. Glucose transporters transport only free glucose

Reaction 2: Isomerization of glucose-6-phosphate to fructose 6-phosphate. The aldose sugar is converted into the keto isoform.

Enzyme: phosphoglucoisomerase.

This is a reversible reaction. The fructose-6-phosphate is quickly consumed and the forward reaction is favored.

Reaction 3: is another kinase reaction. Phosphorylation of the hydroxyl group on C1 forming fructose-1,6- bisphosphate.
Enzyme: phosphofructokinase. This allosteric enzyme regulates the pace of glycolysis (rate limiting step).
ATP is used
Second irreversible reaction of the glycolytic pathway.

Reaction 4: fructose-1,6-bisphosphate splits into 2 3-carbon molecules, one aldehyde and one ketone: dihyroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP).
The enzyme is aldolase.

Reaction 5: DHAP and GAP are isomers and can readily inter-convert by the enzyme triose-phosphate isomerase.
GAP is a substrate for the next step in glycolysis so all of the DHAP is converted to GAP. So, 2 molecules of GAP are formed from each molecule of glucose

Up to this step 2 ATP is used
Second phase: Payoff phase
2 GAP molecules generated from each glucose, therefore each of the remaining reactions occur twice for each glucose molecule being oxidized.

Reaction 6: GAP is dehydrogenated by the enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). In the process, NAD+ is reduced to NADH + H+ from NAD. Oxidation is coupled to the phosphorylation of the C1
carbon.

1,3-bisphosphoglycerate is formed

Reaction 7: This high energy bond of BPG at C-1 is hydrolyzed to a carboxylic acid and the energy released is used to generate ATP from ADP.

Enzyme:phosphoglycerate kinase.

Product: 3-phosphoglycerate.

Reaction 8: The phosphate group shifts from C3 to C2 to form 2-phosphoglycerate.

Enzyme:phosphoglycerate mutase.

Reaction 9: Dehydration reaction catalyzed by enolase (a lyase). A water molecule is removed to form phosphoenolpyruvate which has a double bond between C2 and C3.

Reaction 10: Enolphosphate is a high energy bond. It is hydrolyzed to form the enolic form of pyruvate with the synthesis of ATP. Irreversible step

Enzyme: pyruvate kinase (regulatory enzyme)

Enol pyruvate quickly changes to a more stable keto pyruvate.

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101 Biological Science Inventions and Discoverers

1.       Animal Cell- Theodore Schwann

2.       Plant Cell - Robert Hook

3.       Cell Theory - Schleiden, Schwann and Virchow

4.       Bacteria – Leeuwenhoek

5.       Gram Staining - Hans C Gram

6.       Virus- Dmitri Ivanovsky

7.       Prions- Stanley B. Prusiner (Prions-Infectious proteins)

8.       AIDS virus (HIV)  – Luc Montagnier

9.       Bacteriophages  discovered  by Frederick W. Twort in (1915) & term coined by Félix d’Hérelle  (1917)

10.   Binomial Nomenclature - Carl Linnaeus

11.   Five Kingdom Classification  - Robert H Whittaker

12.   Nucleus- Robert Brown

13.   Lysosome – Christian de Duve

14.   Golgi Complex – Camillo Golgi

15.   Mitochondria – Albert von Kolliker (The mitochondria is a double-membraned cell organelle, known as the powerhouse of the cell which is present in all eukaryotic cells.It was first discovered by Albert von Kolliker in the year 1857. It was named as bioblast by Richard Altman in the year 1886. The term mitochondria was coined by Carl Benda in the year 1898.)

16.   Gene on Chromosome- Walter S Sutton & Bovery. The term gene coined by Johannsen

17.   Chromosomes-  Hofmeister

18.   Cell Division (Mitosis) -Walther Flemming  & Edward Strasburger

19.   Meiosis - Oscar Hertwig (Meiosis was discovered and described for the first time in sea urchin eggs in 1876 by the German biologist Oscar Hertwig.)

20.   DNA Structure -Watson and Crick

21.   Artificial Gene- Har Gobind Khorana

22.   Process of Photosynthesis  - Jan Ingenhousz

23.   Clavin Cycle- Melvin Calvin

24.   Cell Respiration- Adolf Krebs

25.   Antigen, Blood Group – Land Steiner

26.   Autonomic Nervous System - James Langley

27.   Blood Circulation-  William Harvey

28.   Blood Transfusion-  James Blundell

29.   Pencillin – Alexander Fleming (1928)

30.   Insulin – Banting and Best

31.   Chloroform – Harrison and Simpson

32.   DDT – Paul Muller

33.   Stethoscope - Rene Laennec

34.   Heart Transplantation – Christian Barnard

35.   Vaccination, Smallpox Vaccine – Edward Jenner

36.   Louis Pasteur -Treatment of Rabies, Cure of Hydrophobia, Pasteurization

37.   Developed one of the first successful polio vaccines (Injectable) - Jonas Edward Salk

38.   Oral Polio Vaccine– Albert Sabin

39.   X-Ray – Wilhelm Roentgen

40.   ElectroCardioGram (ECG) – Williem Einthoven

41.   Electroencephalography (EEG)  – Hans Berger

42.   PCR (Polymerase Chain Reaction) – Kary B Mullis

43.   Colour Blindness –John Dalton

44.   Discovered that the Malaria parasite is transmitted by mosquitoes - Ronald Ross

45.   Charles Louis Alphonse Laveran  discovered parasitic protozoans as causative agents of infectious diseases such as Malaria and Trypanosomiasis.diseases

46.   Super Bug – Anand Mohan Chakrabarty (Developed  genetically engineered bacteria (Pseudomonas, the oil eating bacteria) using plasmid transfer .)

47.   Microscope - Antonie Van Leeuwenhoek

48.   Compound Microscope  was invented by

49.   Zaccharias Janssen and Hans Janssen

50.   Electron Microscope  was invented by - Knoll and Ruska

51.   DNA Fingerprinting- Sir Alec Jeffreys

52.   RNA interference (RNAi) -Andrew Fire & Craig C. Mello

53.   ATP  Discovery -Karl Lohmann

54.   Enzyme (Zymase) - Edward Buchne

55.   The first artificial heart  Jarvik-7 - Designed by  Willem Johan Kolff and Robert Jarvik.

56.   Genetic Code -Marshall Nirenberg

57.   Six  Kingdom Classification  - Carl Woese

58.   Lamarck - Theory of Inheritance of Acquired Characters (Lamarckism)

59.   Fertilization in Flowering Plants - Strasburger 

60.   Discoveries in relation to Tuberculosis Robert Koch

61.   Diphtheria Antitoxin - Emil von Behring

62.   Vitamins – Hopkins

63.   Antiseptic Surgery - Joseph Lister

64.   Magnetic Resonance Imaging (MRI)-

65.   Paul C. Lauterbur & Sir Peter Mansfield Lister

66.   IVF - Robert Edwards

67.   Chemotheraphy - Paul Ehrlich

68.   CT Scan - Godfrey Hounsfield

69.   Gene Therapy – Anderson

70.   Germ Theory of Disease - Louis Pasteur (Robert Koch isolated specific bacteria that cause TB and Cholera. He discovered the role of antibodies in immune response)

71.   Classical Conditioning - Ivan Pavlov

72.   Principles of Law of Inheritance- Gregor Mendel Oparin and Haldane

73.    Theory of Chemical Evolution of Life -Oparin and Haldane

74.   Discovery of Streptomycin - Selman Waksman -

75.   Mutation Theory - Hugo de Vries

76.   Temin and David Baltimore - Reverse Transcription

77.   Artistole - First System of Classification of Living Things

78.   Charles Darwin - Theory of Evolution by Natural Selection

79.   Barabara Maclintok - Jumping Gene in Maize

80.   First Cloned Mammal(Dolly) - Ian Wilmut

81.   August  Weismann -  Germplasm theory

82.   Green Fluorescent Protein (GFP) - Osamu Shimomura

83.   First Hormone (Secretin) -Ernest Starling and William Bayliss

84.    HeLa First Immortal Cell line -George Otto Gey

85.   (Acetyl Co A) Neurotransmitter - Otto Loewi

86.   DNA  Polymerase - Arthur Kornberg (1956)

87.   First Recombinant (rDNA) molecule - Paul Berg

88.   Law of Minimum -Nitrogen as essential nutrient in Plant - Liebig

89.   The alpha helix and beta sheet in Protein Secondary Structure - Linus Pauling

90.   Discovery of Restriction Enzymes and their application in Molecular Genetics -Arber, Nathans, and Smith

91.    Theory of Chemical Evolution of Life

92.   Concept of 'Ecosystem' - A G Tansley

93.   Chromatography - Mikhail Tswett

94.   Gene Linkage - William Bateson

95.   DNA as Genetic Material - Avery–MacLeod–McCarty

96.   Central Dogma of Molecular Biology- Francis Crick

97.   Gene Reguation Mechanism  in Prokaryotes (Lac Operon) - Jacob and Monod

98.   Boveri–Sutton Chromosome Theory (Chromosome Theory of Inheritance)

99.   Dideoxy DNA Sequencing Method- Maxam–Gilbert and Sanger

100.   Addison's disease - Thomas Addison

101. Ribozyme (Catalytic RNAs)-Sidney Altman and Thomas Cech

102. Gene discovery using Express Sequence Tags (ESTs)- Craig Venter

103.  Molecular Mechanisms of Circadian Rhythm -Dr. Michael Rosbash, Dr. Jeffrey Hall,Dr. Michael Young

104. CRISPR technology - Yoshizumi Ishino 

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