Annotating the Reaction Stages in MACiE
Similarly to the overall annotation, the stage annotation also has two two components:
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
There are four fields that must be completed within ISIS/Base for the stage annotation. These are:
Figure 2, below, shows an example of the stage annotation that is entered using ISIS.
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):
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:
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.
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:
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.
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.
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.
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:
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:
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.