Standard Requirements for Reporting Enzyme Activity Data
Much reported enzyme data is of limited use to others attempting to apply those data, because the conditions under which they obtained are insufficiently documented. This list was compiled, as a service to the community, by the STRENDA Commission to define the minimum amount of information that should accompany any published enzyme activity data.
Level 1, List A:
DATA REQUIRED FOR A COMPLETE DESCRIPTION OF AN EXPERIMENT
The data are required to allow the reproducibility of the results
Version 1.2, June 16th, 2006
| Data |
|
Comments |
| Identity of the enzymes |
|
|
| Enzyme Name, EC-Number |
|
Name, preferably the accepted name from the IUBMB Enzyme List |
| Organism/species & strain |
|
|
| Sequence accession number |
|
|
| Isoenzyme |
|
|
|
|
|
| Additional information on the enzyme |
|
|
| Tissue/organelle |
|
|
| Localization |
|
|
| Post-translational modification |
|
|
|
|
|
| Preparation |
|
|
| Description |
|
e.g., commercial source, procedure used or reference |
| Artificial modificaiton |
|
e.g., truncated, His-tagged, fusion protein, lacking native glycosylation |
| Purity |
|
purity defined by which criteria
e.g., apparently homogenous by PAGE, crude mitochondrial fraction
|
|
|
|
| Assay conditions |
|
|
| Measured reaction |
|
As a stoichiometrically balanced equation |
| Assay temperature |
|
°C |
| Assay pH |
|
|
| Buffer & concentrations |
|
e.g., 100 mM Tris-HCL, 200 mM potassium phosphate |
| Metal salt(s) & concentrations |
|
e.g., 10 mM KCl, 1.0 mM MgSO4 |
| Other assay components |
|
e.g., 1.0 mM EDTA, 1.0 mM dithiothreitol, 0.1 % Triton X-100, coupled assay components |
| Substrates & concentrations |
|
e.g., 100 µM glucose, 5 mM ATP |
| Enzyme/protein concentration |
|
e.g., nmol ml-1 or mg ml-1 |
|
|
|
| Activity |
|
|
| Initial rates of the reaction measured |
|
|
| Proportionality between initial velocity and enzyme concentration |
|
|
| Specific activity |
|
Units necessary:
Expressed as concentration per amount per time, e.g. micromol product formed/(min . mg enzyme protein) - sometimes referred to as enzyme unit or international unit). The catal (mol/second) may alternatively be used as a unit of activity (conversion factor 1 unit = 16.67 nkat).
|
|
|
|
| Methodology |
|
|
| Assay method |
|
a literature reference may suffice for an established procedure that is used without modification |
| Type of assay |
|
e.g., continuous or discontinuous, direct or coupled |
| Reaction of stopping procedure |
|
in the case of discontinuous assays |
| Direction of the assay |
|
e.g., NAD reduction, glucose phosphorylation |
| Reactant determined |
|
e.g., NADH formation, O2 utilization |
| Reaction stoichiometry |
|
e.g., 2 mol substrate oxidized per mol O2 consumed |
|
|
|
| Additional information desirable |
|
|
| Total assay mixture ionic strength |
|
|
| Free metal cation concentrations |
|
e.g., Mg2+ and Ca2+ |
| Reaction equilibrium constant K+ |
|
Define conditions and reaction direction |
This list is also available as pdf file for download (ca. 230 kB)
 top
Level 1, List B
DESCRIPTION OF ENZYME ACTIVITY DATA
The information is required to allow a quality check on the data and
to ensure their value to others
Version 1.2, June 16th, 2006
|
Information required
|
|
Notes/Comments
|
| Data necessary for reporting kinetic parameters |
|
Units necessary |
| Vmax |
|
Vmax given as Units or katal, as defined in List A |
| kcat |
|
Vmax may be diveded by the molar concentration of the enzyme to give kcat, measured in min-1 or s-2 |
| kcat/Km |
|
kcat/Km given as per time per concentration, e.g., s-1.mM-1 |
| Km |
|
Units necessary |
| S0.5 |
|
Both given as concentrations, e.g., mM |
|
|
|
| Hill coefficient, saturation ratio (Rs) etc. |
|
|
| How was the given parameter obtained? |
|
e.g. non-linear curve fitting using least squares, non-parametric method such as direct linear plot, linear regression ot transformed form of rate equation
Note: The use of linear transformations for determining Michealis-Menten parameters is recognised to be inaccurate.
|
| s/Km range used |
|
e.g. 0.1 to 10 |
| Model used to determine the parameters |
|
With explanation of why is the chosen model considered to be the "right" model |
| High-substrate inhibition, if observed, with KI value |
|
|
|
|
|
| Data required for reporting inhibition data |
|
|
| Time-dependenc and reversibility |
|
|
|
|
|
|
For reversible inhibitors:
|
|
e.g. competitive, uncompetitive, etc., |
| Type and KI values |
|
With units and how they were determined |
|
For tight-binding inhibitors:
|
|
Units necessary |
| Association/dissociation rates, inhibition type and KI values |
|
|
|
For irreversible inhibitors: Type
|
|
e.g., non-specific, mechanism-based, "suicide substrate". |
| Appropriate kinetic parameters |
|
There are too many alternative paramters to list here. The reference to a quite comprehensive source is recommended: Enzymes: Irreversible Inhibition. K.F. Tipton In: Nature Encyclopedia of Life Sciences London, http://www.els.net/ [doi:10.1038/npg.els.0000601] (2001). |
| Note: IC50 values |
|
These have been used for both reversible or irreversible inhibition, but the meaning depends on the type of inhibition. |
|
|
|
| Data required for reporting activation data |
|
Similar to the requirements for inhibition data |
|
|
|
| Required information for all enzyme functional data |
|
|
| Number of repeats |
|
Problems of reproducibility? |
| Indication of accuracy |
|
e.g. standard error of the mean, standard deviation, confidence limmits, quartils |
| Specification whether relative to subunit or oligomeric form |
|
|
|
|
|
| Additional material desirable |
|
|
| Kinetic mechanism |
|
e.g. ordered bi-bi |
| Data for cooperative behaviour: model used |
|
Monod-Wyman-Changeux, etc. |
| Time-dependency of enzyme reactions |
|
i.e., duration of initial rate conditions at defined substrate concentrations etc. |
| Example of at least one experiment together with raw data |
|
|
This list is also available as PDF file (ca. 250 kB).
top
Level 2: Organism-related Definitions of Experimental Conditions - preliminary draft
Suggestions which have to be decided by the experts
Conditions
- assay temperature
- assay pH
- buffer & concentrations
- metal salt(s) & concentration(s)
- other assay components & concentrations
- total assay mixture ionic strength
Preparation
- description
- artificial modification
- purity
Extra suggestions
- crowding agents (PEG, proteins, etc.)
- posttranslational modifications
- artificial modifications
top
|
