I can't understand how to study enzyme kinetics. Say I have a lipase and want to study the kinetics of this lipase using a fluorogenic substrate, how would I do this? From what I understand I would use a 96 or so well plate and in each increase the amount of substrate or increase the amount of enzyme and use a fluorescence microscope or a spectrophotometer?


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    $\begingroup$ A basic biochemistry textbook (or Google) will be very useful in answering this. $\endgroup$ – MattDMo May 1 '14 at 14:39

I'm going to try to lay out some basic definitions here in as plain language as I can find.

Its difficult to study enzymes when they are outside the cell, where they may behave quite differently in different contexts. We categorize a given enzyme in its class by kcat, Km and by mechanism (the sort of reaction they catalyze.

kcat is sort of a maximum velocity of the enzyme - when there is a lot of substrate around - what is the rate of catalysis. Ideally there will be zero product present.

Km is sort of the inverse affinity of the enzyme for its substrate, but at half the maximum velocity of the reaction.

The mechanism, which has a couple of definition basically asks how many substrates are bound and in what order, which products are released and in what order, and any intermediates in the reaction which might appear, and in what order.

e.g.: A -> A* -> B* -> B A + B -> C + D

I say 'sort of' because you might never see an enzyme operate at maximum rate in living cells. Substrates are usually rate controlled there - they only have enough of any given compound as they need.

The context that all of these numbers and designations are used is often taxonomical - that is to say you can categorize and sort, and describe what the enzyme does. In practice large differences in the numbers can describe how useful one enzyme is over another, but it doesn't necessarily tell you how an enzyme will definitely behave in vivo or entirely how it will work in an industrial application. Its just a start.


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