This page was produced as an assignment for an undergraduate course at Davidson College.

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 enzyme catalyzing 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
Reaction Catalyzed
Typical
EC
Enzyme
Numb
Name
er
1
Oxidation or Reduction Reactions: The transfer of O, H, or e- Dehydrogenas
atoms and particles from one molecule to another.
e, Oxidase
2
Functional Group Transfer Reactions: The functional group Transferase,
may be methyl-, acyl-, amino-, or a phosphate group
Kinase
3
Hydrolysis Reactions: Cleaving a molecule by adding H2O
Lipase,
Amylase,
Peptidase
4
Non-hydrolytic Cleaving Reactions: Cleaving a molecule with Decarboxylase
a C-C, C-N, C-O, or C-S bond
, Lyase
5
Isomerization Reactions: Intramolecular rearrangement
Isomerase,
Mutase
6
Synthesis Reactions: Making new C-O, C-S, C-N or C-C
Synthetase
bonds by breaking down ATP
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.
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 and reverse 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, the Nomenclature 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 the systemic 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 called molecular docking uses a computer to determine which substrates are good
candidates for a particular enzyme after the enzyme's 3D shape has been determined by X-Ray crystallography. 3D
structures of many proteins are available at the Protein Data Bank online. 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 of amylosucrase (EC 2.4.1.4) with its substrate, sucrose:

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.
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, the fourth EC number is specific to a particular reaction.
First E.C.
Number
1:
Oxidoreductase
s
2: Transferases

Second E.C. Number Means:

Third E.C. Number Means:

Indicates the H+ or e- donor that

undergoes oxidation

Indicates the acceptor

Indicates the group transferred

Further information on the group

transferred
3: Hydrolases
Indicates the nature of the bond
Indicates the nature of the
hydrolysed
substrate
4: Lyases
Indicates the nature of the broken bond
Further information on the
eliminated group
5: Isomerases
Indicates the type of isomerism
Indicates the type of substrates
6: Ligases
Indicates the type of bond formed
Only used in C-N ligases
To assign an EC number to an enzyme that has just been characterized, the chart at the bottom of this page is
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 is then broken into two
separate molecules. We would then click on the EC 3 button by the word Hydrolase in the chart, pictured below:

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 that an 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
BRENDA - The Comprehensive
Enzyme System

What it shows on each enzyme's page

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 the enzyme.
ExPASy - Expert Protein Analysis
Provides links to other databases, including
System
UniProtKB/Swiss-Prot entries for the enzyme in
different species. Each species-specific enzyme page
links to a consensus sequence, a feature table viewer
of the protein, and links to published literature about
the enzyme in that species.
KEGG - Kyoto Encyclopedia of Shows a detailed reaction mechanism, and complete
Genes and Genomes
diagrams of the pathways the enzyme is involved in
ERGO Light - Integrated
Presents the pathway the enzyme is involved in with a
Genomics
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: 775779. Available <http://www.nature.com/nature/journal/v448/n7155/full/nature05981.html>. Accessed 11 November
2008.
NC-IUBMB. n.d. "Classification and Nomenclature of Enzymes by the Reactions they Catalyze". Available
<http://www.chem.qmul.ac.uk/iubmb/enzyme/rules.html>. Accessed 9 November 2008.
NC-IUBMB. 2008. "Enzyme Nomenclature." Available
<http://www.chem.qmul.ac.uk/iubmb/enzyme/index.html#recommend>. Accessed 9 November 2008.
Wikipedia. 2008. "EC Number." Available <http://en.wikipedia.org/wiki/EC_number>. Accessed 9 November 2008.

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