Study of 3.alpha.,20.beta.-hydroxysteroid dehydrogenase with an enzyme-generated affinity alkylator: dual enzyme activity at a single active site

Publisher Website
Google Scholar
Abstract
The substrate 17.beta.-[(1S)-1-hydroxy-2-propynyl]-androst-4-en-3-one (.beta.-HPA) and its enzyme-generated alkylating product 17.beta.-(1-oxo-2-propynyl)androst-4-en-3-one (OPA) were synthesized to investigate the relationship between the 3.alpha. and 20.beta. activities observed in commercially available cortisone reductase (EC 1.1.1.53) from Streptomyces hydrogenans. .beta.-HPA, a substrate [apparent Km = 145 .mu.M; Vmax = 63 nmol (min .mu.g)-1], when enzymatically oxidized by cortisone reductase to OPA, inactivates simultaneously the 3.alpha. and 20.beta. activities in a time-dependent and irreversible manner following pseudo-first-order kinetics. OPA alone, an affinity alkylating steroid (KI = 40.5 .mu.M; k3 = 1.8 .times. 10-2 s-1), simultaneously inactivates 3.alpha. and 20.beta. activities in a time-dependent and irreversible manner. At pH 7 the t1/2 of enzyme inactivation for .beta.-HPA (10 h) or OPA (41 min) is slower than at pH 9.2 (.beta.-HPA, 16 min and OPA, 3.3 min). Substrates (progesterone, 20.beta.-hydroxypregn-4-en-3-one and 5.alpha.-di-hydrotestosterone), but not all steroids (20.alpha.-.DELTA.4-pregn-4-en-3-one and 17.beta.-estradiol) protect against loss of both enzyme activities by .beta.-HPA and OPA. The .alpha. isomer of HPA is not enzymatically oxidized and does not cause inactivation of either 3.alpha. or 20.beta. activity. .beta.-HPA functions as a substrate for the enzymatic generation of a powerful affinity alkylator of cortisone reductase. The identical change in both the 3.alpha. and 20.beta. activities in all experimental conditions results from dual enzyme activity at a single enzyme active site.

This publication has 16 references indexed in Scilit: