Affinity Labeling of Steroid Binding Sites: SYNTHESIS OF 16α-BROMOACETOXYPROGESTERONE AND ITS USE FOR AFFINITY LABELING OF 20β-HYDROXYSTEROID DEHYDROGENASE

Abstract

Abstract To extend the use of affinity labeling for the characterization of macromolecular steroid binding sites, we have synthesized 16α-bromoacetoxyprogesterone and studied its reactions with various amino acids and 20β-hydroxysteroid dehydrogenase from Streptomyces hydrogenans. This compound alkylates cysteine, histidine, methionine, lysine, and β-mercaptoethanol under physiological conditions. Progesterone derivatives containing 16α-bromoacetoxy, 16α-chloroacetoxy, and 16α-acetoxy substituents are substrates of 20β-hydroxysteroid dehydrogenase with apparent Km values of 1.45 x 10-4, 0.11 x 10-4, and 1.25 x 10-4 m, respectively. Under similar conditions progesterone and cortisone have respective values of 3.95 x 10-6 and 5.1 x 10-5 m. Of the three progesterone esters employed, only 16α-bromoacetoxyprogesterone inactivates the enzyme. It does so in a time-dependent, irreversible manner. When 16α-bromoacetoxyprogesterone is present in 100-fold molar excess, the rate of inactivation follows pseudo-first order kinetics with t½ = 3 hours. Progesterone, cortisone, NAD+, and NADH slow the rate of enzyme inactivation. Excess β-mercaptoethanol stops it but does not restore enzyme activity. Tritiated 16α-bromoacetoxyprogesterone, prepared by condensation of 16α-hydroxyprogesterone with [2-3H]bromoacetic acid in the presence of dicyclohexylcarbodiimide, was used to radiolabel the enzyme-active site during inactivation. The amount of radioactivity incorporated during 30, 50, and 80% enzyme inactivation, indicated that in all cases 2 moles of steroid are bound per mole of enzyme. Amino acid analysis of the hydrolysates of 50 and 80% inactivated enzyme revealed that most of the radioactive material (70% of total counts per min) corresponded to known 1,3-dicarboxymethylhistidine. Further, the radioactivity in this enzyme hydrolysate cocrystallizes with authentic 1,3-dicarboxymethylhistidine. These observations are compatible with a mechanism whereby the progesterone moiety of 16α-bromoacetoxyprogesterone delivers the alkylating group to the active site of 20β-hydroxysteroid dehydrogenase, where it reacts with a histidine residue at that site. They provide the first direct evidence for the presence of this amino acid at a dehydrogenase catalytic site. Furthermore, this supports the view that histidine probably participates in dehydrogenation. Finally, 16α-bromoacetoxyprogesterone is offered as a compound which may be used to characterize certain steroid binding sites of high affinity present in receptor proteins of target organs.