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 1.5, October 9th, 2008
| Data |
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Comments |
| Identity of the enzymes |
|
|
| Name of Reaction Catalyst |
|
Name, preferably the accepted name from the IUBMB Enzyme List |
| EC-Number |
|
|
| Sequence accession number |
|
|
| Organism/species & strain |
|
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| Isoenzyme |
|
|
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|
|
| Additional information on the enzyme |
|
|
| Tissue/organelle |
|
|
| Localization |
|
Within cell or experiment? Specify what localization is based on |
| Post-translational modification |
|
Add only when determined |
|
|
|
| Preparation |
|
|
| Description |
|
e.g., commercial source, procedure used or reference |
| Artificial modificaiton |
|
e.g., truncated, His-tagged, fusion protein, lacking native glycosylation |
| Enyzme or protein purity |
|
purity defined by which criteria. Specify whether protein or enzyme was purified
e.g., apparently homogenous by PAGE, crude mitochondrial fraction, determined by MS
|
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|
|
| Assay conditions |
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|
| Measured reaction |
|
as a stoichiometrically balanced equation |
| Assay temperature |
|
°C |
| Assay pressure |
|
if is is not atmospheric; indicate if not aerobic |
| Assay pH |
|
description of confirmation |
| 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 |
| Coupled assay components |
|
if relevant |
| Substrates & concentration ranges |
|
e.g., 1 - 100 mM glucose, 5 mM ATP |
| Enzyme/protein concentration |
|
molar concentration if number of active sites known, otherwise mass concentration
e.g., nmol ml-1 or mg ml-1 or better. µmol l-1 or g l-1
|
| Variable components |
|
|
| Total assay mixture ionic strength |
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| Activity |
|
|
| Initial rates of the reaction measured |
|
Determine how established |
| Proportionality between initial velocity and enzyme concentration |
|
if available |
| Specific activity |
|
Units necessary:
Expressed as amount product formed per amount enzyme/protein present - sometimes referred to as enzyme unit or international unit (1 U = 1 µmol min-1). The katal (mol/s) may alternatively be used as a unit of activity (conversion factor 1 unit = 16.67 nkat).
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| Methodology |
|
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| 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 |
|
With respect to the reaction equation provided
e.g., NAD reduction by alcohol dehydrogenase;
alcohol + NAD+ --> aldehyde or ketone + NADH + H+
|
| 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., of Mg2+ and Ca2+ |
| Reaction equilibrium constant K |
|
Define conditions and reaction direction |
This list is also available as pdf file for download (ca. 252 kB)
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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 1.5, October 9th, 2008
|
Information required
|
|
Notes/Comments
|
| Required data for all functional enzyme data |
|
|
| Number of independent experiments |
|
Problems of reproducibility? |
| Indication of accuracy |
|
e.g., standard error of the mean, standard deviation, confidence limits, quartiles |
| Specification whether relative to subunit or oligomeric form |
|
|
|
|
|
| Data necessary for reporting kinetic parameters |
|
Units necessary |
| Vmax |
|
Vmax given as units or katal, as defined in List 1A |
| kcat |
|
Vmax may be diveded by the specific activity units (moles per unit time per unit enzyme mass) of the enyzme to give kcat, measured in s-2 or min-1 |
| kcat/Km |
|
kcat/Km given as per time per concentration, e.g., s-1.mM-1 |
| Km |
|
Units necessary |
| S0.5 |
|
Both are concentrations, e.g., mM |
| Hill coefficient, saturation ratio (Rs) or other coefficients of co-operativity |
|
|
| 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 |
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| Data required for reporting inhibition data |
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| Time-dependenc and reversibility |
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With method described |
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For reversible inhibitors:
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|
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| Type and KI values |
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With units and how they were determined
e.g., competitive, uncompetitive, etc., |
|
For tight-binding inhibitors:
|
|
Units necessary |
| Association/dissociation rates, inhibition type and KI values
|
|
|
|
For irreversible inhibitors: type
|
|
. |
| Appropriate kinetic parameters |
|
e.g., non-specific, mechanism-based, "suicide substrate"
There are too many alternative paramters to list here. The reference to a quite comprehensive source is recommended: Tipton, K.F. (2001) Enzymes: Irreversible Inhibition. In: Nature Encyclopedia of Life Sciences. London, http://www.els.net/ [doi:10.1038/npg.els.0000601]
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|
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Note: IC50 values
These have been used for both reversible or irreversible inhibition. However, the use is not recommended because these values are without a consistent meaning. The relationship of these values to inhibition constants is analysed in detals, e.g., Cortes, A. et al. (2001) Biochem. J. 357:263-268.
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| Data required for reporting activation data |
|
Similar to the requirements for inhibition data |
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| Additional material desirable |
|
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| Kinetic mechanism |
|
e.g. ordered bi-bi |
| Data for cooperative behaviour: model used |
|
With equation given
e.g., 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. 256 kB).
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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
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