Question

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

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

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

Answer 1

A) We can measure the disappearance of Z at 340 nm (absorbance will decrease), or we can measure the increase in Y at 480 nm (absorbance will increase). This will not measure the enzyme, but will measure the enzyme activity.
B) Turnover number = V_max/E_t

V_max is the maximum velocity and E_t is total amount of enzyme
V_max = 60 µ mol/min

E_t = 0.4mL x 0.1mg/mL = 0.04 mg enzyme    (400 µL = 0.4 mL)
Turnover number = (60 µmol/min) /(0.04 mg)
Turnover number =1500 µmol/min/mg