It basically reflects the bias of an investigator. Like if someone is good in chemistry he would use M/s representation while an enzymologist/biologist will prefer IU. Vm is the maximum velocity that a fixed conc. Of enzyme display when it is fully saturated with a substrate under a given set of experimental conditions. It doesn't talk about the volume of enzyme used. It simply gives rate of the enzyme catalysed reaction (change in substrate conc per unit time). However the rate will definitely be influenced by conc of enzyme. So enzymologists relate it to per unit volume as IU/ml and refer to it as enzyme activity. It is a very helpful parameter during enzyme purification process where after each purification step it must go up ( enzyme activity).

Hope this clarify some doubts

Mohammad Asif Shah Thank you! It does shed light on a few confusions. In that case, kcat would have the unit of s-1 or min-1 in unison. I overlooked the 1/mL part, no wonder I got stuck when explaining the results in some literature.

Indeed, it's been taught that the reaction velocity is based on the substrate concentration until they reach saturation. Since we use 4-MU as a reporter by fluorescence to derive concentrations, it is much more convenient expressing them in mM/s for us.

It is important to distinguish between the two different enzymological meanings of the term unit (U) with respect to catalysis. It can mean micromoles of product formed/minute, but it can also be given an arbitrary, assay-specific meaning. For example, here is the unit definition for the restriction enzyme EcoR1 from New Engalnd Biolabs:

"One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl."

An international unit (IU) is not typically a measurement of enzyme activity, but of the amount of some substance, such as a vitamin, based on a measurement in some biological assay.

https://en.wikipedia.org/wiki/International_unit

The unit of catalytic activity in the SI system is the katal, which is moles of product formed/second.

https://en.wikipedia.org/wiki/Katal

As an Enzymologist, I have come across different units for Vmax. I think this depends first on whether the concentration of enzyme involved in the reaction is known or not. Some report mM/s (for known conc) while some report micromole/ml/min taking into consideration the volume of enzyme used if the concentration is not known.

In any case, this should not be equated with the enzyme activity unit which does not take into account the saturation of the enzyme involved, though having almost the same unit annotation. The specific activity is better used to explain the standard activity of an enzyme by dividing the Enzyme Activity by protein concentration especially during Enzyme purification process.

Does that depend on the purity of enzymes? If one is pure, we show them in enzyme units, otherwise simply M.s-1 altogether for the ease of knowledge communication?

I don't think the purity of the enzyme is relevant to this choice.

Adam B Shapiro Thanks. IU has been no longer recommended for enzyme activity than for nutrition since early 2000s by skimming articles.

kcat (s-1) is the Parameter to use to describe the Enzyme activity.

Vmax is used when you do not know the Enzyme concentration/purity and then it can be described as mole Substrate converted per time unit and per mg or ml Enzyme preparation.

For a description see for example: Guidelines for reporting of biocatalytic reactions https://doi.org/10.1016/j.tibtech.2010.01.001

The enzyme unit for Vmax is EU/ ml. The information of kinetic properties can be understood from Km expression of the affinity of the enzyme and the Vmax catalytic activity of the enzyme from Michael Mentein equation.

The enzyme unit is the amount of the enzyme that converts of 1 Micro mole / ml of substrate per minute to product at 25 centidegrade at optimum condition. According to international unit is M/s.

Practically I used for enzyme purification EU/ml and I purified the enzyme and draw the correlation EU /ml activity of the enzyme with PH, Temperature and so on.

VMAX definition has been a source confusion for a long time, and I think this derives from many textbooks, where the subject of Enzyme Kinetcs is treated superficially. The IUPAC and the IUMBB define enzyme activity in mass of product converted per unit of time by a fixed amount of enzyme.

Thus, the International Unit is µmol/min while the SI unit is the katal (mol/s)

Now this may be confusing, because usually the experiment is run in a liquid assay of certain volume, and the reference or standard curve is run in the same volume, but labelled as "nmol of substance" added to that volume, let us say, 1 ml. When the actual reads from the enzymatic assay are interpolated or converted, the volume of the cuvette is left behind and you end up with an amount of mass as a function of time, i.e. a progress curve, from which, an initial velocity is calculated.

The analysis of the saturation isoterm by a substrate, using several assays, run with the fixed amount of enzyme added to an assay of fixed volume, at variable concentration of substrate, renders a value of Vmax, which is actually not a true kinetic parameter, but the product of

kCAT ET

where kCAT is the pseudofirst order rate constant for the catalytic step or turnover number and represents the number of molecule of substrate converted to product by 1 active site per unit of time;

while ET is the total amount (in number of mols and not as concentration) of enzyme used.

Now, as mentioned by Anders O Magnusson , the true kinetic constant is the kCAT. But, in order to calculate it, the number of mols of enzyme added to the assay is required, and this implies a pure enzyme of know molecular mass, or a preparation where the molar number of active sites was previously titrated, by some method.

Notice the Vmax is an intensive quantity, rather than the extensive value of change in concentration against time and depends on the amount of enzyme added, which is not included in the value. For that reason, when reporting data for an enzyme preparation with an unknown concentration of enzyme active sites, it has become customary to report the Maximum specific activity, that is the VMAX divided by the amount of protein added to the assays. Unfortunately, many authors report this value as VMAX and you will see many tables in professional works where the VMAX is reported in µmol min-1 (mg of protein)-1.

I hope this helps to clarify the issue.

Best wishes

Rogelio

you can look at this definition in:

https://febs.onlinelibrary.wiley.com/doi/abs/10.1111/j.1432-1033.1979.tb13116.x

and

http://www.iupac.org/publications/pac/2001/pdf/7306x0927.pdf

You may also wish to look at the threads:

https://www.researchgate.net/post/Which_is_the_right_unit_for_enzyme_activity&usg=AOvVaw1fR-VOaOL2cGgyW_PPIlaL

https://www.researchgate.net/post/How_can_I_calculate_the_unit_of_enzymes&usg=AOvVaw2LTjr-koUDUxN8bc3j3R4p

Guidelines for describing the results of enzymatic measurements in publications have been promulgated by STRENDA:

https://www.beilstein-strenda-db.org/strenda/public/guidelines.xhtml

Thanks Adam B Shapiro ,

Unfortunately, the guidelines propagate the confusion by asking to report the Maximum specific activity as VMAX. This would be fine is textbooks of Enzyme Kinetics would agree to redefine VMAX as kCAT ET / [Protein]. But that has not been the case.

Best wishes,

Rogelio

"Kind-of-quantities" lit me up. Rogelio Rodríguez-Sotres May I ask some kind-of-stupid questions? If Et is an amount, then Vmax shall be in enzyme units; if Et in molar, then Vmax must be in EU/ml or M/s? Those look really arbitrary, but they somewhat imply the same thing, in my view. Often I come across the symbol [E]0...

I've also seen them in μmol.min-1.[mg-1 protein] if they did not get the pure protein.

Dear Peng-Yuan Chen ,

Questions cannot be stupid.

As you said in the equation VMAX = kCAT ET ,

ET should be put in as mol, mmol, µmol, etc.

If you use [E] concentration, then you get the Maximum "catalytic-activity concentration".

Now be careful, if you use the final concentration in the assay cuvette, you will get the value of the "catalytic-activity concentration" in the final assay. If you use the concentration of the enzyme in the stock used as source for the experiments, you will get the "catalytic-activity concentration" of the enzyme preparation. The last one will equal the former times the dilution factor.

Best wishes,

Rogelio

Check this document as reference:

http://publications.iupac.org/pac/2001/pdf/7306x0927.pdf

Thank you for the guidelines for presenting data from Prof. Anders O Magnusson and for clearing up difficulties by Prof. Rogelio Rodríguez-Sotres .

All together from above, it looks quite united and safe, maybe, to display kcat, KM and kcat/KM only, let alone inhibitor assays.

Yes.

Dear Peng,

Yeah, you are right!! Every manuscript describing enzyme kinetics uses a different unit to describe the significant values such as Km and Vmax. Prof. Rogelio's explanations are spot on.

Peng-Yuan Chen what is the title of the books and articles talking about the variety of expression? and pages numbers please.

HOUTHAIL ALAHMAD ALJAMMAS I'm afraid I have lost track of reading materials. The confusion arose from my revisiting the theory. Because it was more straightforward applying μM/min than μmol/min since that's how people conduct experiments--a container, buffers, enzymes, substrates & a clock/stopwatch--So I googled the keyword "Vmax unit", and then...well, depending on from which perspective to see.😌

I learned it and was taught in Chapter 6 of the 5th edition of Lehninger Principles of Biochemistry, where Km is expressed in μM/min [(μmol/ℓ)/min].