How to Determine EC Numbers_2

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How to Determine EC Numbers

Enzyme Classification (EC) numbers are assigned to chemical reactions catalyzed by

enzymes. Most EC numbers are associated with a name that should refer to the enzymecatalyzing the reaction called a systemic name. Combining the EC number with the systemic

name to describe one protein allows for greater consistency across organisms and clarity

across scientific disciplines.

How An Enzyme's Name Is Determined

EC numbers come in the following form: "EC #.#.#.#". Each level within the EC number

denotes some part of the enzyme's function. The first number splits all catalyzed reactions

into 6 major groups:

First

EC

Number

Reaction CatalyzedTypical

Enzyme Name

1

Oxidation or Reduction Reactions: The transfer

of O, H, or e- atoms and particles from one

molecule to another.

Dehydrogenase,

Oxidase

2

Functional Group Transfer Reactions: The

functional group may be methyl-, acyl-, amino-,or a phosphate group

Transferase,

Kinase

3Hydrolysis Reactions: Cleaving a molecule by

adding H2O

Lipase,

Amylase,

Peptidase

4Non-hydrolytic Cleaving Reactions: Cleaving a

molecule with a C-C, C-N, C-O, or C-S bond

Decarboxylase,

Lyase

5Isomerization Reactions: Intramolecular

rearrangement

Isomerase,

Mutase

6

Synthesis Reactions: Making new C-O, C-S, C-

N or C-C bonds by breaking down ATP Synthetase

After the first EC number division, classifying enzyme reactions becomes trickier. For

example, 1.1.#.# enzymes are oxidoreductases that act on CH-OH groups only. 1.2.#.#

enzymes are those that act on aldehydes or ketones. 1.3.#.# act on CH-CH groups. Within the

1.1 classification, 1.1.1.# enzymes are oxidoreductases that act on CH-OH groups with

NAD+ or NADP+ as receptors. 1.1.2.# enzymes are those that act with cytochrome as the

acceptor, 1.1.3.# means that oxygen is the acceptor, 1.1.4.# means disulfide is the acceptor,

1.1.5.# means that quinone is the acceptor, and he 1.1.99.# designation indicates that

something else is the acceptor.


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This system of nomenclature has some problems, the main one being that an enzyme cannot

be classified or named until the reaction it catalyzes is clear. Also, more than one strand of

amino acids can catalyze the same chemical reaction, and thus have the same EC number and

name. Also, enzymes may catalyze a sequence of different reactions instead of one reaction,

thus complicating the classification and naming process. More than one enzyme may work in

concert to perform a single reaction. Finally, many enzymes perform both the forward andreverse of a catalytic reaction, and thus a standard procedure should be used to determine

which name to use.

To deal with the issue of more than one enzyme catalyzing one reaction, theNomenclature

Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB)

suggests that if more than one enzyme is involved, the word "system" should be appended to

the end of the name. NC-IUBMB also suggests that for enzymes catalyzing a series of

reactions, its name should be assigned based on the first reaction in series, and its following

reactions can be listed in parenthesis after the systemic name. Finally, to deal with the

problem of forward or reverse reactions, the NC-IUBMB recommends that scientists refer to

their volume, which lists all chemical reactions known to be catalyzed by enzymes, and thesystemic name reflects the direction in which the reaction is written. If a scientist discovers a

new reaction, he or she can pick out the systemic name.

How An Enzyme's Function Is Determined

Determining an enzyme's function is a difficult process. Proteins with obvious known

orthologs (similar amino acid chains, conserved binding domains) in different species lend

themselves to function determination easily. However, in proteins with no known orthologs,

scientists must start from scratch to determine an enzyme's substrate and function. A

relatively new method calledmolecular dockinguses a computer to determine which

substrates are good candidates for a particular enzyme after the enzyme's 3D shape has been

determined byX-Ray crystallography. 3D structures of many proteins are available at the

Protein Data Bankonline. Scientists can then use the protein's 3D structures paired with

possible substrate's 3D structures and run thousands of substrates through a computer

program which scores each substrate based on how well it fit into the enzyme's active site.

The following is an image from the Protein Data Bank ofamylosucrase(EC 2.4.1.4) with its

substrate, sucrose:
http://www.chem.qmul.ac.uk/iubmb/enzyme/rules.htmlhttp://www.chem.qmul.ac.uk/iubmb/enzyme/rules.htmlhttp://www.chem.qmul.ac.uk/iubmb/enzyme/rules.htmlhttp://www.chem.qmul.ac.uk/iubmb/enzyme/rules.htmlhttp://www.nature.com/nature/journal/v448/n7155/full/nature05981.htmlhttp://www.nature.com/nature/journal/v448/n7155/full/nature05981.htmlhttp://www.nature.com/nature/journal/v448/n7155/full/nature05981.htmlhttp://en.wikipedia.org/wiki/X-ray_crystallographyhttp://en.wikipedia.org/wiki/X-ray_crystallographyhttp://en.wikipedia.org/wiki/X-ray_crystallographyhttp://www.rcsb.org/pdb/home/home.dohttp://www.rcsb.org/pdb/home/home.dohttp://en.wikipedia.org/wiki/Amylosucrasehttp://en.wikipedia.org/wiki/Amylosucrasehttp://en.wikipedia.org/wiki/Amylosucrasehttp://en.wikipedia.org/wiki/Amylosucrasehttp://www.rcsb.org/pdb/home/home.dohttp://en.wikipedia.org/wiki/X-ray_crystallographyhttp://www.nature.com/nature/journal/v448/n7155/full/nature05981.htmlhttp://www.chem.qmul.ac.uk/iubmb/enzyme/rules.htmlhttp://www.chem.qmul.ac.uk/iubmb/enzyme/rules.html


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The program then lists the potential substrates so the scientists can physically test the top-

scoring substrates and find catalytic rate constants. The scientists can also find the actual

structure of the substrate-enzyme complex via X-ray crystallography and see how closely it

matches the computer prediction. Before the molecular docking method and computer

program was developed, scientists would physically test thousands of substrates, and

chemically determine the products and catalytic rate constants of all of these reactions. The

catalytic rate constant is a measure of how many substrates were changed into product per

unit time (in amylosucrase, this is determined by how fast the enzyme can convert sucrose

and (1,4-alpha-D-glucosyl)n to D-fructose and (1,4-alpha-D-glucosyl)n+1). Once one knows

which reactions an enzyme can catalyze quickly, one can infer the enzyme's function.


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How An Enzyme's EC Number Is Assigned

After one knows the enzyme's function, one can easily determine its EC number and systemic

name by looking at the atoms that move from one species to another in the chemical reaction.

The table above details how to find the first EC number. Once you know the first EC number,

the table below helps figure out what the second and third EC number mean. In all cases, thefourth EC number is specific to a particular reaction.

First E.C.

NumberSecond E.C. Number Means:

Third E.C. Number

Means:

1:

Oxidoreductases

Indicates the H+ or e- donor that

undergoes oxidationIndicates the acceptor

2: Transferases Indicates the group transferredFurther information on the

group transferred

3: Hydrolases Indicates the nature of the bondhydrolysed Indicates the nature of thesubstrate

4: LyasesIndicates the nature of the

broken bond

Further information on the

eliminated group

5: Isomerases Indicates the type of isomerismIndicates the type of

substrates

6: LigasesIndicates the type of bond

formedOnly used in C-N ligases

To assign an EC number to an enzyme that has just been characterized, the chart at thebottom ofthis pageis helpful. Let's say we found an enzyme that catalyzed this reaction:

trithionate + H2O = thiosulfate + sulfate + 2 H+

We can say that this enzyme is a hydrolase because it adds H20 into the molecule, which isthen broken into two separate molecules. We would then click on the EC 3 button by the

word Hydrolase in the chart, pictured below:
http://www.chem.qmul.ac.uk/iubmb/enzyme/index.html#recommend%00http://www.chem.qmul.ac.uk/iubmb/enzyme/index.html#recommend%00http://www.chem.qmul.ac.uk/iubmb/enzyme/index.html#recommend%00http://www.chem.qmul.ac.uk/iubmb/enzyme/index.html#recommend%00


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From the images of trithionate, the substrate, and thiosulfate and sulfate, the products, we can

see that in this reaction, a sulfur-sulfur bond is cleaved. This leads us to believe that our

reaction is denoted by the EC number 3.12. Click on the "separate" button that is on the same

row as EC 3.12: Acting on sulfur-sulfure bonds, as pictured below:

If you follow the links through 3.12.1 and 3.12.1.1 (the only options available), you find thatan enzyme catalyzing this reaction is called "trithionate hydrolase" with EC number 3.12.1.1.

To figure out more about this EC number, you can look at these sites:

Website What it shows on each enzyme's page

BRENDA- The

Comprehensive Enzyme

System

Each EC entry has links to other databases,

easily available information about which

organisms the enzyme has been found in, the

inhibitors and activators of the enzyme, data

about the enzyme's optimal conditions and

physical structure, and links to literature about
http://www.brenda-enzymes.org/index.php4http://www.brenda-enzymes.org/index.php4http://www.brenda-enzymes.org/index.php4


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the enzyme.

ExPASy- Expert Protein

Analysis System

Provides links to other databases, including

UniProtKB/Swiss-Protentries for the enzyme

in different species. Each species-specific

enzyme page links to a consensus sequence, afeature table viewer of the protein, and links to

published literature about the enzyme in that

species.

KEGG- Kyoto Encyclopedia

of Genes and Genomes

Shows a detailed reaction mechanism, and

complete diagrams of the pathways the enzyme

is involved in

ERGO Light- Integrated

Genomics

Presents the pathway the enzyme is involved in

with a list, and a searchable database of EC

numbers found in all organisms so you can find

which organism has your enzyme.

Now you know how to find what an unknown enzyme does, determine its EC number, and

use the EC number to find out more about the enzyme. You also know how systemic names

are produced, and the rules scientists follow to create the systemic names and assign EC

numbers. The information and databases presented will allow scientists to share discoveries

about completely new enzymes or previously discovered enzymes in a new species.

References

Hermann, et al. 2007. "Structure-based Activity Prediction for an enzyme of unkown

function". Nature 448: 775-779. Available

. Accessed 11

November 2008.

NC-IUBMB. n.d. "Classification and Nomenclature of Enzymes by the Reactions they

Catalyze". Available . Accessed 9

November 2008.

NC-IUBMB. 2008. "Enzyme Nomenclature." Available

. Accessed 9

November 2008.

Wikipedia. 2008. "EC Number." Available .

Accessed 9 November 2008.
http://www.expasy.org/http://www.expasy.org/http://www.uniprot.org/http://www.uniprot.org/http://www.genome.ad.jp/kegg/kegg2.htmlhttp://www.genome.ad.jp/kegg/kegg2.htmlhttp://www.ergo-light.com/ERGO/CGI/index.cgi?org=http://www.ergo-light.com/ERGO/CGI/index.cgi?org=http://www.ergo-light.com/ERGO/CGI/index.cgi?org=http://www.genome.ad.jp/kegg/kegg2.htmlhttp://www.uniprot.org/http://www.expasy.org/

How to Determine EC Numbers_2

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