Affinity Labeling of Steroid Binding Sites

Abstract

To extend the use of affinity labeling for the characterization of macromolecular steroid binding sites, we have synthesized 16a-bromoacetoxyprogesterone and studied its reactions with various amino acids and PO@-hydroxysteroid dehydrogenase from Streptomyces hydrogenans.This compound alkylates cysteine, histidine, methionine, lysine, and &mercaptoethanol under physiological conditions.Progesterone derivatives containing 16a-bromoacetoxy, 16archloroacetoxy, and 16a-acetoxy substituents are substrates of 20P-hydroxysteroid dehydrogenase with apparent K,,, values of 1.45 X lo-*, 0.11 X lo-*, and 1.25 x lo-*M, respectively.Under similar conditions progesterone and cortisone have respective values of 3.95 X 10m6 and 5.1 X 10V6 M.Of the three progesterone esters employed, only 16abromoacetoxyprogesterone inactivates the enzyme.It does so in a time-dependent, irreversible manner.When 16c~bromoacetoxyprogesterone is present in loo-fold molar excess, the rate of inactivation follows pseudo-first order kinetics with t+ = 3 hours.Progesterone, cortisone, NAD+, and NADH slow the rate of enzyme inactivation.Excess &mercaptoethanol stops it but does not restore enzyme activity.Tritiated 16ar-bromoacetoxyprogesterone, prepared by condensation of 16ac-hydroxyprogesterone with [2-aH]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