Enzyme X has a molecular weight of 48,000. It converts substrate Z into product

Question

Enzyme X has a molecular weight of 48,000. It converts substrate Z into product Y. Z absobs at 340nm, and Y absorbs at 480nm.

1. At what wavelength do you measure the change in absorbance to assay for enzyme X? Does the absorbance increase or decrease over time?

2. If Vmax is 60micromoles/min and you use 400 microliters of a 0.1 mg/mL solution of enzyme, what is the turnover number?

3.  Why is the vmax not a constant? Why do we want to analyze Kcat instead of Vmax?

Please show work so I can understand how you got the answers.

Explanation / Answer

1)

we will measue at 480nm ....because

specific activity of enzyme = (moles of producted formed / min /mg od substrate )

product formation increases with time

so absorbance increases with time

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2)

Kcat = Vmax /Total enzyme

convert enzyme in to moles

1 ml ----------------> 0.1 mg

400 *10^(-3) ml --------------------?   

contains = 0.04 mg

moles = 8.33 E(-7)

Kcat = 60 E(-6 )/ 8.33 E(-7 )

= 72 /min

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3)

Vmax is dependent on the enzyme concentration and substrate concentration, which changes during the time course.

kcat is independent of any concentration and so is a constant.

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