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Enzyme Kinetics - Turnover

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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Enzyme Kinetics - Turnover

Postby TJC12 » Sat Feb 25, 2012 9:35 pm

Hi! :)

I am having a few problems calculating some required enzyme kinetic values. I've investigated the activity over 3 minutes of alcohol dehydrogenase at varying [S]. I used a spectrophotometer for this. From each of those graphs i've calculated the gradient (A/min) and converted this using the beer-lambert law and the reaction volume to give me velocity in "moles of product formed/min". I used this to then plot a lineweaver burke plot to get Vmax and Km values.

One of the questions asks me to calculate the Kcat value. This is where i'm stuck. I have the following data at hand:

Vmax = 2.442 x 10E-2 μmoles/min
Amount of enzyme used each time = 0.05mL
Concentration of supplied enzyme = 4μg/mL
Molecular Weight of Enzyme = 150,000 Mr

The enzyme however exists as a homotetramer, and has 4 identical active sites. I know I need to take that into consideration but i'm generally confused. Could someone talk me through step by step please? :) Thanks!

- TJC
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Postby JackBean » Sun Feb 26, 2012 9:47 am

I'm not sure now, is K_cat taken per enzyme molecule or per active site?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby TJC12 » Sun Feb 26, 2012 12:19 pm

I am also unclear as to it's definition, i've seen it defined either way in numerous places. However the question i've been given does specifically make a point that it's a homotetramer and asks for the Kcat/turnover number per active site.
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