Hi there,

You favorite enzyme might be prone to oligomerization and this process might be inhibiting its activity. Wen diluting the equilibrium is displaced towards monomeric form which might be the more active form. An other explanation might be the presence of an inhibiting molecule within your enzyme prep. By diluting the enzyme you also dilue the inhibitor.

Hi there,

You favorite enzyme might be prone to oligomerization and this process might be inhibiting its activity. Wen diluting the equilibrium is displaced towards monomeric form which might be the more active form. An other explanation might be the presence of an inhibiting molecule within your enzyme prep. By diluting the enzyme you also dilue the inhibitor.

Hi Dominique,

Thank you for your answer. 

I tried the measurement by standard protocol and by using a kit (Glutamate dehydrogenase kit from sigma) and I found the same results. So, I think that there is no inhibiting molecules and perhaps this is due to the oligomerization process. 

Could you recommend me any papers or articles regarding this?

Do you follow the activity in time? Do you calculate the activity in Units/molar (specific activity?). It might be that at a fixed time points, substrate gets depleted and it seems as if activity is less (per molar enzyme). 

Hi Joost,

Thank you

Yes, I followed it in time. I calculated it in IU/mg Enzyme (GDH). When I used the kit, I calculated it in milliunits/ml (as recommanded by sigma). And for both of them I just took the slope for the measurement. Does it make any sens?

I have been encountered to the similar phenomena in assaying the rat kidney- and brain- biotinidase, too.   Other rat organs and rat serum do not show this type of the dilution effect at all.   Therefore, the result of diluted states (a graph showing the variation of initial velocity and protein concentration) in kidney and brain must have been adopted; i.e., we are the first to demonstrate the Amo (= kcat/Km) of biotinidase in the brain is highest (though the 9th organ of V in the rat; file J Chrom B,,).   The first is left cerebrum, 2nd is testis, and 3rd is kidney in the male rat, and 1st is left cerebrum, 2nd is kidney, and 3rd is large intestine in the female rat, respectively.   The reason may be hydrophobic interactions among hydrophobic enzyme and other hydrophobic membrane components (glycolipids (gangliosides), phospholipids, steroids, and membrane glycoproteins) of kidney and brain.  Since hydrophobicity of  the mitochondrial glutamate dehydrogenase is 0.542, which is higher than the human serum biotinidase of 0.535.   Mitochondrial enzyme has no glycochain, but human serum biotinidase has it.   Then, hydrophobicity is more linked to the aggregation phenomena rather than glycochain; i.e., glycochain' s major role is the controlling of the enzyme kinetics.

Human serum biotidase is dimer enzyme, but V is not changes in response to dilution.   Inhibitor will diluted according to dilution, but inhibition rate should not be changed; i.e., inhibitor is effective by binding to enzyme strongly, and ratio of inhibitor and enzyme will not be changed by simple dilution (addition of non-ionic detergent is more important; file surfactant BIN).   Addition of surfactant may release the hydrophobic inhibitors (such as leucine in glutamate dehydrogenase) and membrane glycoproteins attached to hydrophobic enzyme of serum biotinidase.   However, surfactant has not been used in the rat paper (file J Chrom B rat BIN LIP) in order to precisely compare the organ differences.

Therefore, I am very grateful to Dr. Ahlem Zaghmi (National de la Recherche Scientifique, Institute Armand-Frappier Research Centre, Québec, Canada), who has helped me to arrive at the awareness about the essential role of glycochain attached to the human serum biotinidase.  

Thus, the sufficient dilution is performable only by using a sensitive HPLC-enzyme assay (file J Chrom B,,,).

Further, it is noteworthy that skin's enzyme is difficult to handle with; i.e., collagen induces aggregation (from sol to gel) of the skin homogenate during standing on ice, and homogenized samples should have been prepared many times.   Then, HPLC-SEC protein assay is very convenient, since analysis time is rapid  within 3.5 min per assay (file HPLC-SEC,,,).   

The first answer by D. Liger gives the two mechanisms that may explain your experimental data. In the second the inhibitor may be the substrate. You will find more information on such mechanisms is a recent review.

www.ncbi.nlm.nih.gov/pmc/articles/PMC3298769/pdf/nihms344914.pdf

The first mechanism is discussed on p. 9, your enzyme given after the extensive literature list.

This link is to a paper on an another enzyme where the same is observed as in your experimental data.

www.jbc.org/content/266/12/7682.full.pdf