| Human prostatic steroid 5alpha-reductase catalyzes the irreversible conversion of testosterone to dihydrotestosterone with NADPH as cofactor. In this study, three different mutant cDNAs were reconstructed: the A49T missense cDNA (a constitutional mutation), G183D cDNA (a double somatic coexisting mutant that occurs with A49T in prostate cancer) and A49T-G183D double mutant cDNA. To optimize the in vitro experiments, several variables (pH, time, protein amount) were examined before the substrate Vmax value was measured. The Vmax and substrate K m of wild type SRD5A2 are 1.17 nmole/min*mg and 0.92 muM respectively; whereas those of the A49T mutants are 2.62 nmole/min*mg and 7.13 muM, which seems to be an inherent gain-of-function because of the higher Vmax. These enzyme activities may contribute to future studies of steroid 5alpha-reductase inhibitors in human patients treated for prostatic conditions. |
