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Topic: Is our Alkaline Phosphatase's Km value is high or low?  (Read 1022 times)

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Offline Random Particle

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Is our Alkaline Phosphatase's Km value is high or low?
« on: January 11, 2019, 03:12:01 PM »
Hello :)

We've been testing the enzyme Alkaline Phosphatase kinnetics using different concentrations of the enzyme. based on our results, we found out that our enzyme's Km is 0.831 mM.
we know that a small Km indicates high affinity to the substrate and that a large Km means low affinity. We couldn't find out if our value is high or low, then we came across an article which stated that p-Nitrophenyl phosphate's Km is 1.5 mM.
Does the value from the article states the optimal Km value? Does it means that our value is low and with strong affinity to the substrate?

Thanks in advance!

Offline Babcock_Hall

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Re: Is our Alkaline Phosphatase's Km value is high or low?
« Reply #1 on: January 11, 2019, 06:21:35 PM »
One ordinarily varies the concentration of substrate, not enzyme, in order to determine the KM.  The value of KM is expected to vary with pH and might vary with ionic strength.  With alkaline phosphatase, the choice of buffer can also influence the values of the kinetic constants because of an additional complication: the buffer Tris, for example, is an alternate acceptor of the phosphoryl group (the normal acceptor is water).  One would have to see the article in order to evaluate the solution conditions.

It is not always true that the KM is a measure of affinity between enzyme and substrate in the same way that the dissociation constant is.  The KM may or may not be equal to Kd; it depends on the enzyme and the substrate in question.

Offline Random Particle

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Re: Is our Alkaline Phosphatase's Km value is high or low?
« Reply #2 on: January 12, 2019, 03:16:47 AM »
Im sorry, forgot to mention that we worked with buffer tris on pH=9 the whole time. Our substrat was p-Nitrophenyl phosphate. They where diluted in water.
We already did an experiment with different substrat concentrations but now we have been asked, how different concentrations of the enzyme affects the kinetics.
The reaction accured at same conditions all the time except the enzyme's concentrations. We couldn't find articles that explains the actual values of the Km and their meanings.

We suppous to explain the meaning of Km value (0.831) that we got. But we don't know if this value indicates a small or high Km.

Thanks again!

Offline Babcock_Hall

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Re: Is our Alkaline Phosphatase's Km value is high or low?
« Reply #3 on: January 12, 2019, 09:12:07 AM »
The KM is higher than expected, but you might use the BRENDA database or PubMed to search more.

You may have encountered something called the turnover number, given the symbol kcat.  By definition Vmax = (kcat)•[Etotal].  Try rewriting the Michaelis-Menten equation substituting this expression in for Vmax, and see if this helps.

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