v = Vmaxy(1 + (Vmax2[S]/Vmax;K,)) / (1 + Km/[S] + [S]/K,) (4)

 Values for K, and Vmax derived from equation 1 were used as initial values in the

fitting of data to equations 3 and 4. Eadie-Hofstee plots were used in order to analyze the

biphasic kinetics observed.

 Results

Sulfonation and glucuronidation of 3-OH-B[a]P

 Optimum conditions for sulfonation were 10 minutes incubation time and 25 Cpg

cytosolic protein. A concentration of 0.02 mM PAPS provided saturating concentrations

of the co-substrate and enabled kinetic parameters at 1.0 CLM 3-OH-B [a]P to be calculated

by the application of eq. 1 (Table 3-la). The data for the sulfonation of 3-OH-B[a]P was

fit to a two-substrate model (eq. 3), whereby the binding of a second substrate to the

enzyme is responsible for the steep decline in enzyme activity at concentrations

exceeding 1 CLM (Figure 3-2a). Initial estimates of Vmaxi and Km were provided by the

initial data obtained at low [S] (non-inhibitory), while Vmax2 WAS constrained to 65 + 20

pmol/min/mg, which is slightly below the plateau in Figure 3-2a.

 The kinetic scheme (Figure 3-2b) illustrates the proposed partial substrate

inhibition process, which assumes that substrate binding is at equilibrium, which is

probable due to the low turnover rate of SULT. The best fit of the data was provided by a

K, of 1.0 + 0.1 CLM. Binding of the second substrate molecule results in a tenfold

reduction in the rate of sulfonate formation.