The substrate 17β- [(1S)-1-hydroxy-2-propynyl]-androst-4-en-3-one (β-HPA) and its enzyme-generated alkylating product 17β-(1-oxo-2-propynyl)androst-4-en-3-one (OPA) were synthesized to investigate the relationship between the 3α and 20β activities observed in commercially available cortisone reductase (EC 18.104.22.168) from Streptomyces hydrogenans.β-HPA, a substrate [apparent Km = 145 μM; Vmax = 63 nmol (min μg)-1], when enzymatically oxidized by cortisone reductase to OPA, inactivates simultaneously the 3α and 20β activities in a time-dependent and irreversible manner following pseudo-first-order kinetics. OPA alone, an affinity alkylating steroid (K1 = 40.5 μM; K3 = 1.8 × 10-2 s-1), simultaneously inactivates 3α and 20β activities in a time-dependent and irreversible manner. At pH 7, the t1/2 of enzyme inactivation for β-HPA (10 h) or OPA (41 min) is slower than at pH 9.2 (β-HPA, 16 min, and OPA, 3.3 min). Substrates (progesterone, 20β-hydroxypregn-4-en-3-one, and 5α-dihydrotestosterone), but not all steroids (20α-Δ4-pregn-4-en-3-one and 17β-estradiol), protect against loss of both enzyme activities by β-HPA and OPA. The a isomer of HPA is not enzymatically oxidized and therefore does not cause inactivation of either 3α or 20β activity. Thus, β-HPA functions as a substrate for the enzymatic generation of a powerful affinity alkylator of cortisone reductase. Second, the identical change in both the 3α and 20β activities in all experimental conditions clearly results from dual enzyme activity at a single enzyme active site.