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Why is km important in mutant enzymes?


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#1 Tired

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Posted 15 April 2009 - 03:31 PM

Hi all,

I have a question that's been bothering me all day. I have generated a library of mutant enzymes and obtained kinetic data (km to be precise) from them. The km of a particular substrate against 2 of my mutants gives a km of 6mM whereas against the other 2 mutants I have a km of >30mM. The same substrate against the WT gives me a km of 6mM. What i don't understand is that why is it important to know the km of a substrate against mutant enzymes when I already know the km towards the wild type?? I mean do these numbers really mean anything??? ;)

Thanks in advance for any suggestions!

BTW, these mutants are single amino acid substitutions.

#2 T C

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Posted 15 April 2009 - 08:16 PM

Hey,

Don't remember it very well but I think this is what it means:

It depends upon the context actually. If you talk about Km of the mutant enzymes that means that you are comparing the affinity of the mutant enzyme for the substrate as compared to the wild type protein. Km basically tells you about how good the substrate is. The lower the Km, higher is the affinity and better is the substrate. This means that at a lower concentration of the substrate, the enzyme reaches half its maximum velocity.

Kcat is the turnover, if you wana compare the turnover of the enzymes. The enzyme having higher kcat is better.

and kcat/Km tells you about the efficiency of the enzyme, the higher the kcat/km, the more efficient is the enzyme.

Hope it helps. But do check textbooks as well.

Best,
TC

Hi all,

I have a question that's been bothering me all day. I have generated a library of mutant enzymes and obtained kinetic data (km to be precise) from them. The km of a particular substrate against 2 of my mutants gives a km of 6mM whereas against the other 2 mutants I have a km of >30mM. The same substrate against the WT gives me a km of 6mM. What i don't understand is that why is it important to know the km of a substrate against mutant enzymes when I already know the km towards the wild type?? I mean do these numbers really mean anything??? :(

Thanks in advance for any suggestions!

BTW, these mutants are single amino acid substitutions.



#3 DRT

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Posted 15 April 2009 - 09:50 PM

Hi all,

I have a question that's been bothering me all day. I have generated a library of mutant enzymes and obtained kinetic data (km to be precise) from them. The km of a particular substrate against 2 of my mutants gives a km of 6mM whereas against the other 2 mutants I have a km of >30mM. The same substrate against the WT gives me a km of 6mM. What i don't understand is that why is it important to know the km of a substrate against mutant enzymes when I already know the km towards the wild type?? I mean do these numbers really mean anything??? :(

Thanks in advance for any suggestions!

BTW, these mutants are single amino acid substitutions.



Perhaps your question should be turned around to read "Why mutate enzymes?" :)

#4 sharath

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Posted 21 April 2009 - 02:30 PM

From your experiment it appears that the single amino acid subsitution represents an active site residue that particiaptes in substarte binding. Kinetics of mutants need to be analysed to establish the effect of mutations on the enzyme function.
Sharath B.




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