TABLE 1.
Properties of native huPGHS-2 and S530A huPGHS-2
Specific activities were determined using a standard O2 electrode assay with 100 μM AA as substrate. Km values were determined using AA concentrations from 1 to 100 μM. The percentage of inhibition by diclofenac was determined in a standard O2 electrode assay with 100 μM AA after a 10-min preincubation of the enzymes at 24°C with 10 μM diclofenac versus without diclofenac; 10 μM diclofenac was also added to the assay chamber in the case of enzyme pretreated with diclofenac. Activation of activity by 20:1ω9 was measured using 5 μM AA plus 25 μM 20:1ω9 in the assay mixtures. Activation of activity by palmitic acid (16:0) was measured using 5 μM AA plus 25 μM 16:0 in the assay mixtures. The rates reported are maximum rates occurring after a lag phase. Igor Pro version 6.0 was used for graphing rates versus AA concentrations in obtaining Km and Vmax values. Errors are reported as standard deviations from at least three kinetic measurements for each substrate or inhibitor concentration.
| Property | huPGHS-2 |
|
|---|---|---|
| Native | S530A/S530A | |
| Specific activity with AA (U/mg protein = μmol O2/min/mg protein) | 40 ± 1 | 14 ± 1 |
| Km with AA (μM) | 5 ± 1 | 9 ± 1 |
| Inhibition by diclofenac (%) | 100 | 15 ± 1 |
| Stimulation by 20:1ω9 (%) | 132 ± 5 | 135 ± 7 |
| Stimulation by 16:0 (%) | 176 ± 5 | 155 ± 11 |