Acetazolamide (AZA) is a widely-used drug in the treatment of glaucoma. It acts

Question

Acetazolamide (AZA) is a widely-used drug in the treatment of glaucoma. It acts by inhibiting carbonic anhydrase II, thereby reducing pressure in the affected eyes. The following initial rate data reaction were obtained using 0.0025 Mu/mL of carbonic anhydrase (M_1 = 29,100 Da): Plot these data in a Michaelis plot. label both axes, including units Determine K_M (mM) and V_max. (mM/min) for both curves. Calculate the turnover number for the uninhibitaed enzyme (in units of sec^-1). What type of inhabitor is AZA

Explanation / Answer

CO2 + H2O-------------------> H2CO3 (the enzyme that catalyses this reaction is carbonic anhydrase)

b.

For the reaction without the inhibitor,

Vmax= 5.21 mM/min

Km = [S] when Vo= 1/2 Vmax; here Vmax = 5.21 mM/min; hence 1/2 Vmax = 2.6 mM/min.

[S] at Vo= 2.6 mM/min can be obtained from the curve- that will give the Km (it will be close to 8 mM)

For the reaction with the inhibitor,

Vmax= 3.92 mM/min

Km = [S] when Vo= 1/2 Vmax; here Vmax = 3.92 mM/min; hence 1/2 Vmax = 1.96 mM/min.

[S] at Vo= 1.96 mM/min can be obtained from the curve- that will give the Km (it will be close to 16 mM)

c. For the uninhibited reaction, Vmax = kcat X [E total] where kcat gives the turnover number.

Vmax= 5.21 mM/min = 0.086 mM/ sec = 8.6 X 10 -5 M/sec

E total = 0.0025 ug/mL = 0.0025 mg/L = 2.5 X 10 -6 gm/ L

The molecular weight of 29100 Da means 29100 gm/ mole.

Hence, 8.6 X 10 -5 M/sec =  kcat X ( 2.5 X 10 -6 / 29100) M

or,  kcat =1001040 sec -1

d. AZA is a non-competitive inhibitor.

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