Computational Systems Biology

Computational Systems Biology

Lecture 2: Enzymes
with the contribution of Luna De Ferrari

Friday 13 January 2006

Images from: David L. Nelson, Lehninger Principles of Biochemistry, IV Edition, W. H. Freeman ed.

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Summary:
Chemical reactions Enzymes Metabolic Pathways Proteins regulating proteins
(phosphorylation, transcriptional regulation and signal transduction)

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Chemical reactions

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Atoms and bonds

Chemical bonds connect the atoms in molecules A chemical bond contains energy
(needs energy to be created and releases energy when is broken)

Images from: David L. Nelson, Lehninger Principles of Biochemistry, IV Edition, W. H. Freeman ed.

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Chemical reactions
A chemical reaction transforms one or more reactants (molecule type) in one or more products (another molecule type) A reaction normally breaks existing bonds and generates new bonds

Example of chemical reaction: N2 + 3 H2 2 NH3

That is: nitrogen (found most often in nature in a two atom molecule N reacting with hydrogen (again a two atom molecule H to form ammonia H N H H N) H)

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The Equilibrium Constant

Reactants and products
aq = in acqeous (water) solution m, n and p are the number of molecules involved in each reaction (stochiometric coefficients)

The concentration of reactants and products at equilibrium defines the equilibrium constant The free energy change G of a chemical reaction depends on the equilibrium constant Keq
(where R is a constant and T is the temperature see next slide)
From: http://en.wikipedia.org/wiki/Equilibrium_Constant http://en.wikipedia.org/wiki/Gibbs_free_energy

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from: David L. Nelson, Lehninger Principles of Biochemistry, IV Edition, W. H. Freeman ed.

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The Equilibrium Constant

for N2 + 3 H2
2 NH3 For the title reaction: A is N2, B is H2, C is NH3 and there is no product D. Also: k=1, m=3 and n = 2 that is 1
molecule of N2 reacts with 3 molecules of type H2 to give 3 molecules of ammonia 1 N2 + 3 H2 2 NH3

And the equilibrium constant K becomes: 2

[NH3] K= 3 [N2] [H2]

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Free energy change

The free energy change G ( = at standard pressure/temperature/pH) of a reaction tells us whether the reaction needs energy to happen or it releases energy (like in the case below where the product P has less energy than the substrate S)
P = product (e.g. NH3), S = substrate / reactant (e.g. H2), = transition state

Images from: David L. Nelson, Lehninger Principles of Biochemistry, IV Edition, W. H. Freeman ed.

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Enzymes

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Enzymes
Most enzymes are proteins, and their activities depends on the 3D structure of the amino acids that compose them

Images from: David L. Nelson, Lehninger Principles of Biochemistry, IV Edition, W. H. Freeman ed.

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The role of enzymes in reactions

IMPORTANT: enzymes do NOT and cannot affect the equilibrium / free energy difference of a reaction (this depends only on the internal energy/bonds of the Substrates and Products species) Enzymes effect is to enhance the reaction rates (molecules produced per second) by lowering the activation energy of the transition state Gcat

Images from: David L. Nelson, Lehninger Principles of Biochemistry, IV Edition, W. H. Freeman ed.

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Transition state energy changed by an enzyme

(in blue)

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The enzyme action on the transition state

Images from: David L. Nelson, Lehninger Principles of Biochemistry, IV Edition, W. H. Freeman ed.

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The enzyme active site

The red molecule is the substrate, the active site appears painted in red

From: David L. Nelson, Lehninger Principles of Biochemistry, IV Edition, W. H. Freeman ed.

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PDB: Protein Database Bank

For other 3D structures:
The protein database bank: http://pdbbeta.rcsb.org/pdb/Welcome.do or The molecule of the month at: http://www.rcsb.org/pdb/static.do?p=education_discussion/m olecule_of_the_month/index.html

From: David L. Nelson, Lehninger Principles of Biochemistry, IV Edition, W. H. Freeman ed.

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Enzyme:

The surface inside the active site

You can see a flash demo of this enzymatic reaction on: http://bcs.whfreeman.com/lehninger/ clicking on Chapter 6: Enzymes mechanism animation fig 6.21 mechanism for chymotrypsin

Images from: David L. Nelson, Lehninger Principles of Biochemistry, IV Edition, W. H. Freeman ed.

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E.C. Enzyme nomenclature

Enzymes are named depending on the reaction they catalyse. Examples of enzyme groups are:
EC 1 - Oxidoreductases EC 2 - Transferases EC 3 - Hydrolases EC 4 - Lyases EC 5 - Isomerases EC 6 - Ligases

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An example of E.C. enzyme nomenclature

EC 3. Hydrolases
EC 3.1 Hydrolases Acting on Ester Bonds EC 3.1.3 Phosphoric Monoester Hydrolases
EC 3.1.3.1 alkaline phosphatase EC 3.1.3.2 acid phosphatase EC 3.1.3.3 phosphoserine phosphatase EC 3.1.3.4 phosphatidate phosphatase EC 3.1.3.5 5'-nucleotidase EC 3.1.3.6 3'-nucleotidase EC 3.1.3.7 3'(2'),5'-bisphosphate nucleotidase EC 3.1.3.8 3-phytase EC 3.1.3.9 glucose-6-phosphatase EC 3.1.3.10 glucose-1-phosphatase

EC 3.1.3.11 fructose-bisphosphatase

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fructoseExample: fructose-bisphosphatase EC 3.1.3.11

From the IUBMB (International Union of Biochemistry and Molecular Biology) web site: http://www.chem.qmul.ac.uk/iubmb/enzyme/ one can reach a record for the enzyme: EC 3.1.3.11 (fructose-bisphosphatase): http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/1/3/11.ht ml The site also contains the reaction diagram shown here: http://www.chem.qmul.ac.uk/iubmb/enzyme/reaction/polys acc/Calvin2.html
The reaction involves the hydrolysis (separation) of a phosphate group here represented by Pi The P representing the phosphate group that appears on the top region of the D-fructose-1,6-biphosphate substrate molecule has disappeared after the reaction, and the product is now called Dfructose 6-phospate (the phosphate group in position 1 is no more there).

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Metabolic pathways

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The KEGG Pathway Database

A metabolic pathway is a chain of enzymatic reactions. The pathway is a collection of step by step
modifications: the initial substance used as substrate by the first enzyme is transformed into a product that will be the substrate for the next reaction, until the exact chemical structure necessary for the cell is reached

http://en.wikipedia.org/wiki/Metabolic_pathway

Pathway databases, like KEGG can be queried online:

http://www.genome.ad.jp/kegg/pathway.html

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A map example from KEGG

Map from: http://www.genome.ad.jp/kegg/pathway/map/map00051.html

fructose-bisphosphatase (EC 3.1.3.11)

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Proteins regulating other proteins:

Phosphorylation

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Phosphorylation
Many proteins/enzymes are regulated inside the cell by the addition of a highly energetic phosphate group (PO4) to one (or more) of the aminoacids on their surface: this process is called phosphorylation. The phoshpate addition alters the functional shape of the enzyme, physically locking it in an activated (or inactivated) form until the phosphate group is removed Hence, two important classes of enzymes are: Kinases they add a phosphate group to another protein/enzyme Phosphatases they remove a phosphate group from a protein/enzyme Kinases and phosphatases are highly specific in their choice of substrate and often a kinase will act only on a single, particular kind of substrate protein. And the substrate protein can itself be another kinase, generating a cascade of activations/inactivations through the cell

1. 2.

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A process affected by phosphorilation:

the cell cycle

From top left: if the DNA is damaged the cell should not divide before repairing it. When the DNA is repaired p53 detaches p21 from the CDK2-CyclinE complex. This complex is a kinase. The CDK2-CyclinE kinase phosphorylates pRb. The addition of a phosphate group modifies pRbs shape and affects its ability to bind E2F. E2F is hence released and free to carry out its role: to promote transcription of other enzymes needed for the cell division cycle
Images from: David L. Nelson, Lehninger Principles of Biochemistry, IV Edition, W. H. Freeman ed.

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Proteins regulating other proteins:

Transcriptional regulation

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Regulation at transcription level

Some proteins - transcriptional activators or repressors - bind to regulatory sequences near the promoter region upstream of a gene (or of a group of genes - operon) to regulate the production of the protein(s) coded by that gene(s)

Activators facilitate the binding of RNA polymerase on the promoter, while repressors impede the binding and hence the transcription

Images from: David L. Nelson, Lehninger Principles of Biochemistry, IV Edition, W. H. Freeman ed.

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Transcriptional regulation: the Lactose operon

Image from: Recombinant DNA, Watson

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Proteins regulating other proteins:

Signal transduction

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Characteristics of Biological Signal transduction

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Biological Signal Transducers: proteins

e.g. a kinase domain that phosphorylates other proteins

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Neuronal signalling
Some proteins exposed on the cell surface cross the entire membrane and act as gates The gates (or receptor channels) can open due to a conformation change, following phosphorylation or binding of a small chemical molecule (for example a drug) This is how synapses of neurons fire to help you think