Just to recall the essential information that should always accompany the publication of enzyme-functional data.
Necessary information when publishing
Notes and comments
Recommended name of the enzyme
Name of the organism from which the enzyme originates (preferably the binomial name)
Examples: EC 188.8.131.52 – Glutathione transferase, Mus musculus
EC 184.108.40.206 – ABC-type lipopolysaccharide transporter, Escherichia coli
Loss of enzyme activity during purification and upon storage is a common phenomenon. We must distinguish between the active center concentration, determined by a titration method and the concentration as protein, which can be determined e.g. photometrically using the absorption coefficient, or by a colorimetric method, e.g. the Bradford assay.
This issue is not trivial because the catalytic constant kcat, as V/[E]t (limiting rate divided by the total enzyme concentration), is often calculated without specifying the method used for determining this concentration or using a value, which does not correspond to the active center concentration determined by titration. As a consequence, the literature contains innumerable kcat values that, for the same enzyme and assay conditions, span ranges far larger than those expected from experimental error. So, don’t be afraid to present your data using the limiting rate V in place of kcat since the conclusions will be the same.
In some cases, the ‘kcat-question’ described on the left becomes enigmatic, suggesting that goblins are at work in laboratories to scramble measurements or calculations. Here an example for the cysteine peptidase cathepsin B, EC 220.127.116.11, from Homo sapiens, substrate Cbz-Phe-Arg-7-amino-4-methylcoumarylamide. In three publications (1-3), the following values for kcat (s−1) have been reported: 42.2 (1), 364 (2) and 1500 (3). The enzyme active center concentration was measured with the same irreversible inhibitor and all assays conditions were the same.
Since the rate of enzyme-catalyzed reactions depends on temperature, this is mandatory information. For enzyme kinetics, a precision of ±1°C is fine.
All components and counter-ions with their concentrations, as well any other additives should be specified. Some buffer substances have small temperature coefficients (e.g. for phosphate this is −0.0028 per oC), while others have large temperature coefficients (e.g. for Tris this is −0.028 per oC). Therefore, the temperatures of buffer preparation and that of the assays should always be stated.
Laboratory pH-meters can take into account the temperature at which buffer solutions are prepared, accuracy being ±0.02 pH units and ±0.5°C. It is therefore advisable to show the pH with two decimal places, e.g. pH 7.40. In addition to specifying all species in a solution, its ionic strength should also be reported. Online applications support all necessary calculations.
The policy of preparing concentrated buffers, e.g. 10x, and diluting them 1:10 before assays is not recommended. Depending on the buffer substances, dilution may produce a change of pH, and correcting the pH by adding acid or base will change the ionic strength and composition: not for kinetics!
In publishing enzyme-kinetic results, it is fine to mention the software used. However, the reader will not take advantage of this information unless the procedures are described in due detail. For instance, in case of nonlinear regression, the calculated parameters should be accompanied by the goodness of fit and the results of any runs test performed shown graphically. In particular during the analysis of allosteric modifiers, some parameters con show a high degree of mutual dependency. In this case, specifying any parameter constraints introduced in the regression procedure, showing the covariance matrix and the parameter dependency clarifies any doubt.
Above all, it is important to be aware of the assumptions made when running nonlinear regression (see the list in the column on the right). Such assumptions are important because, if violated, the parameters resulting from the analysis might be erroneous or even meaningless. Changing nonlinear raw data, for which nonlinear regression is the ideal method of analysis, into linear transforms, impairs these assumptions, notably they distort experimental error. Well-known examples are the double-reciprocal plot and the Scatchard plot.
I like very much the following sentence by William Wallace Cleland, pioneer in enzyme kinetics and much more.
… Emphasis should thus be placed on improving precision and repeating experiments a number of times, rather than on more sophisticated techniques for analysis of bad data … And above all, it is important to use your head in making evaluations based on statistical analysis; all mathematics in the world is no substitute for a reasonable amount of common sense. (4, p.138)
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- Illy C, Quraishi O, Wang J, Purisima E, Vernet T, Mort JS (1997) Role of the occluding loop in cathepsin B activity. J Biol Chem 272: 1197-1202. doi:10.1074/jbc.272.2.1197
- Baricos WH, Zhou Y, Mason RW, Barrett AJ (1988) Human kidney cathepsins B and L. Characterization and potential role in degradation of glomerular basement membrane. Biochem J 252: 301-304. doi:10.1042/bj2520301
- Barrett AJ, Kirschke H (1981) Cathepsin B, cathepsin H, and cathepsin L. Meth Enzymol 80: 535-561. doi:10.1016/S0076-6879(81)80043-2
- Cleland WW (1979) Statistical analysis of enzyme kinetic data. Meth Enzymol 63: 103-138.