A genomic study has shown that an enzyme was about 40 times upregulated in the liver. How to estimate the maximum amount of substrate, knowing the its molecular mass?
Hello
You got to do an enzyme assay and calculate Vmax... Then you'll get to know thethe details..
Hi there,
You first need to purify the enzyme to homogeneity and then run enzyme assays in order to determine KM and kcat(working at constant enzyme concentration and variable substrate concentration).
I agree with Dominique. The measurement kcat is the number of molecules of substrate a molecule of the enzyme converts to product per unit time when saturated with its substrate, under a given set of conditions. The actual amount of product the enzyme produces per unit time in vivo is difficult to determine because the substrate concentration is usually not known, and the conditions differ from the in vitro conditions. There may also be allosteric effectors present in vivo, product inhibition, and competing substrates.
Yes Go for enzyme kinetics. Calculate the Km, Vmax and Kcat for your purified enzyme and from Kcat your can find the turnover number or catalytic efficiency of your enzyme protein.
Good wishes,
I thank Drs. Raghu, Dominique, Adam and Ashok for their valuable contributions regarding ADH4 pharmacokinetics.
Sincerery,
Ivanldo Medeiros
Hi Ivanildo,
If I understand rightly, 40 times upregulated in a liver, especially coming from the context of a genomic study, may mean overexpression of the respective mRNA. This may or may not correlate with the turnover, kcat, of the enzyme. Leave aside the activity aspect, the absolute protein levels may themselves not be correlated with the aboslute mRNA levels. I hope this will help you somehow in modeling the pharmacokinetics right.
Dear Dr. Bharath,
His remarks were valuable to me!
Best Regards,
Ivanildo
Hi Ivanildo,
If you have an enzyme with Michaelian behaviour, make diverse assays with several substrates concentration (preferentially more than 10). The Lineweaver-burk plot allows you to determine both Km and Vmax. By determining the enzyme concentration you can calculate the kcat from the Vmax. Remember to ajust the units corretly (i.e. assay time in seconds, substrate and enzyme concentration in the same unit).
Please, do not hesitate to contact me if any further support is demanded.
Best,
Fabio
Dear Dr. Fábio Kendi,
I am very much grateful for your support regarding the ADH issue.
Sincerely,
Ivanildo Coutinho
if you have an enzyme, and you already know its specificity constant or kcat/Km for a given substrate, you can use that substrate in vivo to back calculate the enzyme concentration. Once you know the enzyme concentration, you can calculate the specific activity for any substrate, by using a substrate concentration below the Km and measuring fractional turnover. The way you know you are below the Km, is to use at least two substrate concentrations, and show the kcat/Km doesn't change and the initial velocity is linear with substrate concentration. You don't need to do a full Km. To calculate the enzyme concentration, if you don't have any data on any substrate specificity constants beforehand, you will need to titrate the enzyme with a tight binding inhbitor and fit the data to the Morrison equation. By titration, I mean that the enzyme concentration has to be above the Ki or inhibition constant in the experiment, and one uses increasing amounts of inhibitor till there is complete inhibition. The fractional inhibition curve is what is fit to the Morrison equation.
Dear Dr. Marcia Moss,
Thank you for your invaluable help concerning the enzyme pharmacokinetics.
Best Regardars,
Ivanildo C. Medeiros
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