Annotating the Reaction Stages in MACiE

Similarly to the overall annotation, the stage annotation also has two two components:

1. Stage annotation in ISIS/Base
2. Stage annotation using the annotation script

Note: Every effort should be made to include every step involved in the reaction mechanism. For example, where the reaction is known to proceed via a Schiff Base formation, authors in the primary literature frequently assume knowledge of this step. However, MACiE explicitly includes all the steps involved in this reaction. Whilst this can make for very long reactions (the longest currently being 14 individual steps) it is believed to be important when looking at the similarity of reactions.

Annotation in ISIS/Base

Figure 1: Screen Shot of Stage Reaction in ISIS/Base

There are four fields that must be completed within ISIS/Base for the stage annotation. These are:

1. Step Number. This should be a number from 1 to n, and represents the number of the step.
2. Step Id. This is analogous to the overall reaction Unique Identifier. This identifier is made up from the ID assigned by ISIS, e.g. 1, and enough zeros to make the number four digits in length, e.g. 0001. Finally the text string ".stg" is added to signify that the reaction is a reaction step, and the number of the step with enough zeros to make the number two digits in length, e.g. 01. This identifier will become the file name of the exported step reaction.
3. Reaction. This is the chemical representation of the reaction step. It is created in ISIS/Draw and then exported as described previously (Using ISIS).
4. Step Comment. This is a free text field into which a comment relating to the reaction step might be added. Comments can be anything relevant to the step, e.g. that the mechanism of the step is not clear, alternative mechanisms for a particular step have been suggested, there exist different possible catalytic amino acid residues, etc.

Figure 2, below, shows an example of the stage annotation that is entered using ISIS.

Figure 2: Screen Shot of Partially Completed Stage Reaction in ISIS/Base

Once this annotation has been completed, the remainder should be done using the annotation script, available to registered developers.

Annotation using the Script

This script is only available to registered developers of MACiE and is password protected. Below is a screen shot of the overall annotation script. If you would like to become a registered developer, please email me with details on how you would like to cotribute.

Whilst the overall reaction annotation is relatively simple, the reaction stages annotation is much more complicated, in both content and how the script works. In order to keep the web interface simple, the first page the user encounters is similar to that for the overall annotation with the following fields (see Figure 3):

Figure 3: Screenshot of Initial Script for Annotating the Reaction Stages

Reacting Species

Substrates and Products

All the substrates (chemical species consumed during the course of the reaction) and products (chemical species which is formed during the course of the reaction) that occur in each reaction step should be listed. This includes the following:

• Overall reactants and products. These should be listed by either their KEGG number, or X number, in rare cases, their PDB code.
• Reaction intermediates. These are transient chemical species formed and then used up during the course of the reaction. These should be named in one of the two following ways:
  1. If it has been seen more than once (e.g. the semiquinone form of FAD) it should be given a permanent identifier, e.g. a KEGG number, or if there is no KEGG number, an X number. Before assigning an X number, check to see if it is already present or not.
  2. Otherwise, it should be numbered according to the reaction it is occurring in, e.g. the first (1) reaction intermediate (I) formed in the first MACiE entry (M0001) would be named M0001-I1.
• Reactant Amino Acid Residues. These are defined as any amino acid residue that undergoes a change in either its electronic state or covalent bonding. For more information on amino acid residues, please see the next section. Amino acid residues in the substrate and product lists should be named as follows: The PDB code of the entry, a hyphen, the three letter amino acid residue code in capital letters followed by the residue number e.g. 1b73-CYS178
• Reactant Cofactors. These should only be included in the substrate and product lists if they are involved in that particular step, in a manner analogous to the amino acid residues. They should be named in these fields as follows: The PDB code of the entry, a hyphen, the HET group code (if this is not possible, then the abbreviated cofactor name) followed by the residue number e.g. 1b73-FAD444.

Cofactors Present

A cofactor is a chemical species which is required (but not consumed) by the enzyme in order for the enzyme to be active. When applied to an enzyme reaction step, the term is extended to incorporate any chemical species which is considered a cofactor in the overall reaction.

Cofactors should be listed individually due to the fact that there is further annotation to be included when the first page of annotation is complete. They are named using their HET group code and number in the sequence that is assigned from the PDB file. If cofactors from multiple chains are involved, the chains are differentiated with the chain identifier, again from the PDB file, e.g. FAD444B, FMN445A, where FAD444 is from chain B, and FMN445 is from chain A.

If there are an insufficient number of fields here, please contact me, and I will add more to the script, as further and more detailed annotation of the cofactors is dependent on the fields being correctly, and singly, filled in.

The further annotation required for the cofactors, shown below, is accessed when the first page is updated. It requires the user to select options for two fields.

Figure 4: Second stage of cofactors present annotation

The first field is the Type of cofactor that is named. This is either metal, or non-metal and is included for ease of distinguishing between MACiE annotation, and the Metal-MACiE annotation.

The second field is a list of functions (attributes) performed by the cofactor in that particular step. If the cofactor is not performing any reactant function in a given step, it should be annotated as an onlooker. If it is not performing any function in that particular step, it should have no other annotated function. For a description of the functions, please see the next section and the MACiE glossary.

Amino Acid Residues Present

Amino acid residues should be listed individually due to the fact that there is further annotation to be included when the first page of annotation is complete. They are named using their three letter code and their number in the sequence is assigned from the PDB file. If amino acids from multiple chains are involved, the chains are differentiated with the chain identifier, again from the PDB file, e.g. Glu56B, Asp185A, where Glu56 is from chain B, and Asp185 is from chain A. Due to the re-mediation of the PDB, all chains should now have an identifier.

Where there are residues with multiple active portions, the residue name should include a location identifier:

1. Side Chain
2. Post Translationally Modified
3. Main Chain Amide
4. Main Chain Carbonyl
5. Main Chain C alpha
6. Main Chain C Terminus
7. Main Chain N Terminus

If there are an insufficient number of fields here, please contact me, and I will add more to the script, as further and more detailed annotation of the amino acid residues is dependent on the fields being correctly, and singly, filled in.

The further annotation required for the amino acid residues, shown below, is accessed when the first page is updated. It requires the user to select options for two fields.

Figure 5: Second stage of residues present annotation

The first field is the location of the amino acid residue's function. This can be the side chain, main chain amide, main chain carbonyl, main chain N-terminus, main chain C-terminus or main chain Cα and finally, post-translationally modified residues (those residues that have been modified from their original form). More information on the various portions of amino acid residues can be found in the next section (Amino Acid Residues Involved).

The second field is a list of functions performed by the amino acid residue in that particular step. If the amino acid residue is not performing any reactant function in a given step, it should be annotated as an onlooker. If it is not performing any function in that particular step, it should have no other annotated function. For a description of the functions, please see the next section and the MACiE glossary.

Chemistry Involved

The Reactive Centres should be annotated. These are defined as any atom at which a reaction occurs, such that a bond is either formed, cleaved or altered in order or an atom at which the oxidation state changes. They should be listed as if a unique ID was assignable to each one, thus there may be multiple occurrences of each atom type. Unfortunately, unique ID's are not assignable in ISIS, however, we hope to eventually automate this particular annotation, which will allow unique ID's to become assignable.

The Rate Determining Step? buttons are used to indicate whether the step being annotated is the rate determining step (RDS). This flag is set whenever the step being annotated is the step in a reaction which has the slowest rate, thus imposing an upper limit on the overall reaction rate. There are three options available to the RDS: Yes, No, and Unknown. However, this flag is very rarely set to anything other than unknown (the default) as many references do not explicitly state which step is rate determining. Once this flag has been set there is no further annotation associated with it. Unless there is kinetic data in the literature reference the only rate determining steps that can be assigned are single step reactions. If a step in the reaction has been assigned as the RDS, then all other reaction steps should be listed as not being rate determining.

The Reversible? radio buttons are used to indicate the reversibility of the reaction step. A reversible reaction can be described as a reaction which can proceed in the forward direction as well as in the reverse direction. Most enzyme reactions are reversible and so the default value for this flag is Yes. It is sometimes true that it would be unclear as to whether a reaction is reversible or not, hence the Unknown flag, and finally, enzyme reactions are not always reversible. Once this flag has been set, the user need take no further action on the question of reversibility.
The reversibility flag is more important for the CML version of MACiE as it is only in CML that it becomes possible to fully utilise all that reversibility means. In CML it should be possible to search for reactants on both the reactant and product side of the reaction in a reversible reaction. In ISIS/Base there is no such thing as a reversible reaction, due to the limitations of the MDL definition of a reaction, hence the reversibility flag is the only easy way to define a reaction as being reversible.

Stage Reaction Mechanisms

This is a list of the possible descriptions for the mechanism of the reaction, such as nucleophilic addition. This includes the The Ingold Reaction Mechanisms. More information on these are found in a future section reaction stage mechanisms.

There are certain reaction mechanisms that have further annotation associated with them, which is accessed when the first page is updated. The following mechanisms all have further annotation associated with them:

• Proton, hydrogen, hydride and electron transfers all have transfer from and transfer to. These fields take a list of the chemical species that donate (transfer from) and accept (transfer to) the species being transferred. Where there is more than one species in the chain, the transfer from and transfer to boxes take a comma separated list. E.g. In a proton transfer from species1 to species2 to species3, the transfer from field would take the form species1,species2 and the transfer to field would take the form species2,species3
• Redox reactions also have further annotation, which involved the species oxidised and species reduced fields

Stage Reaction Attributes

This is a list of the overall effects of the reaction, including bonds involved. Further information on these can be found in the section reaction stage attributes.

Similarly to the reaction stage mechanisms, many of the attributes have further annotation associated with them:

• Bonds Formed, Bonds Cleaved and Bonds Polarised. These require a comma separated list of all the bonds formed, cleaved or polarised during the course of the reaction step. They should be included as if a unique identifier were assignable to every bond.
• Bonds Changed in Order. These require a comma separated list of all the bonds changed in order during the course of the reaction step. These are listed in a different manner to the bonds formed or cleaved, they should be listed in the following way: initial species-initial bond, initial bond order, final species-final bond, final bond order. E.g. M0001-I1-C=C, 2, M0001-I2-C-C, 1, M0001-I1-C-N, 1, M0001-I2-C=N, 2
• Intermediate Formation and Collapse. Requires a comma separated list of the intermediates formed and collapsed during the course of the reaction step.
• Charge Delocalisation requires a comma separated list of the atoms over which the charge is delocalised.

Reaction Description

Each step in the reaction mechanism must also have a textual description of the step. This should be succinct and as brief as possible and should detail the important functional roles of the amino acid residues and/or cofactors along with the mechanism occurring. e.g. for M0065.stg01: Asp13 deprotonates IMP with concomitant tautomerisation to give the oxyanion, which is stabilised by a hydrogen bond from Gln224. Activated IMP is produced. His41 and Mg stabilise the GTP. For other examples, please look at other MACiE entries.

Finishing the Stage Annnotation

Once the annotation on this initial page has been completed, click on the Update button which creates the second annotation page. The script uses the values it has been given to determine the fields it requires for the completion of the reaction .

Once the further annotation has been completed, check the Write to File check box and click on update. This will produce the complete stage annotation at the top of the page, which is then copied and pasted into ISIS/Base. Completing the stage annotation, as shown in the figure below.

Figure 6: Screen Shot of Completed Stage Reaction in ISIS/Base