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Topic: Enzyme Kinetics Parameters at different Enzyme titre  (Read 10458 times)

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Offline curiouscat

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Re: Enzyme Kinetics Parameters at different Enzyme titre
« Reply #15 on: March 21, 2016, 01:53:59 PM »
  One thing that is a bit odd; they give the concentration of "proteins" which implies that there is a mixture of proteins and that the concentration of any one enzyme might be less than that.  Just brainstorming here.

I think the "proteins" bit may have an innocuous explanation. They are studying four different enzymes SaSSy / SaBS etc.

So they are referring to those four varied enzymes as "proteins"? So it's not a mixture but just different experiments, one for each "protein".

Offline Yggdrasil

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Re: Enzyme Kinetics Parameters at different Enzyme titre
« Reply #16 on: March 21, 2016, 01:57:50 PM »
Given that the concentration of enzyme is greater than the Km, at low substrate concentrations you will be looking at the pre-steady state kinetics (looking at single turnover events by the enzyme) rather than the steady state conditions (reflecting multiple turnover events) that the Michaelis Menten equation is meant to describe.

Ah! Great point.

So, that would imply it is just a bad measurement strategy employed by the study authors?

i.e. They ought to have taken measurements at way lower [ E0] or way higher [ S]?

Am I interpreting what you wrote correctly? In other words, is this kinetics data I got from the paper usable at all?

Yeah, from the description of the experiments, it seems like the data may not be appropriate for use in a MM analysis.

Can you post the citation for the paper(s), maybe it'll be easier to figure out what's going on with access to the full texts.

That said, it's not uncommon for kinetic parameters for a particular enzyme to differ between studies for varying reasons (for example, the quality of the protein prep can vary between groups).

Offline curiouscat

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Re: Enzyme Kinetics Parameters at different Enzyme titre
« Reply #17 on: March 21, 2016, 01:59:58 PM »
Can you post the citation for the paper(s), maybe it'll be easier to figure out what's going on with access to the full texts.

Sure. Here is a link. The full text is accessible online:

http://www.nature.com/articles/srep10095

A link to the Uniprot database kinetics entry for the enzyme is here:

http://www.uniprot.org/uniprot/E3W202

Offline Babcock_Hall

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Re: Enzyme Kinetics Parameters at different Enzyme titre
« Reply #18 on: March 21, 2016, 09:29:40 PM »
The mystery deepens! I got another set of kinetic constants for the same enzyme from the Uniprot database:

kcat is 0.34 sec(-1)

KM=1.4 µM

These seem quite different from the earlier constants. Unfortunately the [ E0] isn't mentioned here. (What good is kcat & kM is you don't mention a [ E0]? You can't use these constants quantitatively without an [ E0] value, right?)

http://www.uniprot.org/uniprot/E3W202
The kinetic constants Km and kcat should be independent of [ E0] in theory.  My initial posts were meant to stress that the assay has to be validated with respect to changes in [ E0] in practice.

Offline Yggdrasil

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Re: Enzyme Kinetics Parameters at different Enzyme titre
« Reply #19 on: March 22, 2016, 11:03:40 AM »
A link to the Uniprot database kinetics entry for the enzyme is here:

http://www.uniprot.org/uniprot/E3W202

I would trust the numbers in the source cited by Uniprot (http://www.jbc.org/content/286/20/17445.long), as their reaction conditions ("For determination of steady-state enzyme kinetic constants, conditions were as described previously except the enzyme concentration was kept at 10 nm. Substrate concentrations ranged from 1 μm to 100 μm, and reactions were incubated at 30 °C for exactly 5 min.") seem much well suited for determining steady-state reaction kinetics than the Sci Rep paper.

Offline Babcock_Hall

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Re: Enzyme Kinetics Parameters at different Enzyme titre
« Reply #20 on: March 23, 2016, 09:24:19 AM »
I agree.  I would also contact the authors for clarification regarding their concentration of enzyme.

Offline curiouscat

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Re: Enzyme Kinetics Parameters at different Enzyme titre
« Reply #21 on: March 24, 2016, 10:33:03 AM »
I agree.  I would also contact the authors for clarification regarding their concentration of enzyme.

Thanks for your help @Babcock & @Yggdrasil

I think I will do as you suggested. The nM conc. seems the right one indeed!

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