242 Experiment 24-secitrate Detydragense an Enayme of the Citeric Acld Gedle POS

Question

242 Experiment 24-secitrate Detydragense an Enayme of the Citeric Acld Gedle POST-LAB QUESTIONS 1. What kind of enzyme activity would you observe if you did not grind the yeast safficiendly and only 10% of the cells were broken? 2. Isocitrate dehydrogenase has an optimum pH of 7.0. Did you work under optimal condition If so, how did you provide for i? 3. Inspect your plot. Why didn't you take the ahsorbance at 5 min. rather than at 60 se to calculate the enzyme activity? If you would have used the 5 min. value would your enzyme activity be higher, lower or the same as the one you calculatedP Explain

Explanation / Answer

Ans. #1. If yeast sample is not homogenized sufficiently, the observed enzyme activity would be lower than the expected enzyme activity there would be lesser amount of enzyme molecules present in the inefficiently homogenized sample.

#2. Yes.

The optimal pH was provided by adding buffer. Since buffers resists change in pH, presence of buffer in the reaction mixture, or better say, the reaction is carried out in buffered solution, minimizes the deviation from optimal pH during reaction.

[Note: Provide the name of buffer you used or listed in the protocol. It shall possibly be a phosphate buffer].

#3. The enzyme catalysis or reaction begins as soon as the substrate is mixed with the enzyme sample.

Suppose, you take the absorbance of product produced during reaction.

# At time t=0 second, the absorbance of reaction mixture is 0.0.

As soon as the substrate is added to reaction mixture, the concentration of products begin to increase due to catalysis of substrate by enzyme. So, absorbance also increases.

# Note that we measure the change in absorbance at certain intervals, say at interval of 30 seconds or 60 seconds. The change in absorbance gives change in product concentration and thus the rate of enzyme catalysis (or, the rate of formation of product OR rate of substrate catalysis per unit time).

# Rate of enzyme catalysis is generally very high. So, taking absorbance after 5 minutes would virtually lead to no subsequent change in absorbance (say, Abs at 6th minute remains the same as Abs at 5th minute) because almost all the substrates have already been converted into product in first 2-3 minutes of the reaction. That is, nothing change in absorbance of the reaction mixture after 5 minutes would give no result.

Therefore, the absorbance of reaction mixture is taken immediately after adding the substrate but not after 5 minutes.    

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