Problem 2: Cyclophosphamide (CPA) is an anticancer prodrug that requires activat

Question

Problem 2: Cyclophosphamide (CPA) is an anticancer prodrug that requires activation in the liver by cytochrome P450 2B enzymes for production of cytotoxic metabolites. Site-specific mutagenesis is used to alter the amino acid sequence of P450 2B1 in an attempt to improve the kinetics of CPA activation. The rate of reaction of CPA is studied using rat P450 2B1 and a site-specific variant of P450 2B1 produced using Escherichia coli. The results are shown below. Determine the kinetic constants (Vmax and Km) for the rat and site- specific variant enzymes. Initial CPA Concentration 0.0003 0.0005 0.0008 0.0015 0.003 0.005 0.007 Initial Reaction Velocity mol/l.mirn rat P450 2B1 5.82 9.03 12.7 17.1 20.2 27.8 31.5 Initial Reaction Velocity mol/l.min variant P450 2B1 17.5 24.5 24 23.9 27.3 33.1 27.7

Explanation / Answer

Answer

Vmax = 1/b Km = mVmax

Rat enzyme: trendline equation = 0.0423x + 0.0288,

Vmax = 1/0.0288 = 34.72,

Km = 0.0423(34.72) = 1.47

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