Ralph M. Pollack

Irreversible active-site-directed inhibition of Δ5-3-ketosteroid isomerase by steroidal 17-β-oxiranes. Evidence for two modes of binding in steroid-enzyme complexes

The spiro-17β-oxiranyl derivatives of d-equilenin, epiandrosterone, dehydroepiandrosterone, and Δ4-androsten-3,17-dione are active-site-directed irreversible inhibitors of Δ5-3-ketosteroid isomerase of P.testosteroni. The 17β-oxiranyl steroids rapidly inhibit the isomerase in a time-dependent manner which exhibits saturation kinetics. The enzyme is protected against inactivation by the competitive inhibitor 19-nortestosterone. In addition, prolonged dialysis against neutral buffer leads to no regeneration of enzyme activity. Fluorescent spectral changes associated with the incubation of the enzyme with the 17β-oxirane derived from d-equilenin indicate that there are two modes of binding for steroids to the isomerase. These results suggest that 17β-oxiranes may inhibit the enzyme by a mechanism similar to that for the previously studied spiro-3β-oxiranyl steroids.

An active-site-directed irreversible inhibitor of Δ5-3-ketosteroid isomerase

Abstract The α and β isomers of spiro-3-oxiranyl-5α-androstan-17β-ol were tested as possible inhibitors of Δ5-3-ketosteroid isomerase of Pseudomonas testosteroni . The β-oxirane causes a first-order irreversible inactivation of the enzyme and shows saturation kinetics (KI, 17 μM). Protection against inactivation is exhibited by 19-nortestosterone, a competitive inhibitor of the isomerase. Although the α-oxirane was found to be a good reversible inhibitor (Ki, 21 μM), prolonged incubation with it failed to produce any inactivation of the isomerase. The results obtained are consistent with the presence of a nucleophilic group situated near the 3-keto group of the substrate in the enzyme-steroid complex.

Experimental Evidence for Enzyme-Enhanced Coupled Motion/Quantum Mechanical Hydrogen Tunneling by Ketosteroid Isomerase

Although there are considerable data demonstrating that quantum mechanical hydrogen tunneling (HT) occurs in both enzymatic and nonenzymatic systems, little data exist that address the question of whether enzymes enhance the amount of HT relative to the corresponding nonenzymatic reactions. To investigate whether 3-oxo-Delta (5)-steroid isomerase (ketosteroid isomerase, KSI) enhances HT relative to the nonenzymatic (acetate-catalyzed) isomerization of Delta (5)-androstene-3,17-dione ( 1) to Delta (4)-androstene-3,17-dione ( 3), alpha-secondary deuterium kinetic isotope effects (KIE) at C-6 of the steroid were determined for both the KSI- and acetate-catalyzed isomerizations. The normal intrinsic secondary KIE for both wild type (WT) KSI (1.073 +/- 0.023) and acetate (1.031 +/- 0.010) suggest the possibility of coupled motion (CM)/HT in both the enzymatic and nonenzymatic systems. To assess the contribution of CM/HT in these reactions, the secondary KIE were also measured under conditions in which deuterium instead of hydrogen is transferred. The decrease in secondary KIE for WT (1.035 +/- 0.011) indicates the presence of CM/HT in the enzymatic reaction, whereas the acetate reaction shows no change in secondary KIE for deuterium transfer (1.030 +/- 0.009) and therefore no evidence for CM/HT. On the basis of these experiments, we propose that KSI enhances the CM/HT contribution to the rate acceleration over the solution reaction. Active site mutants of KSI (Y14F and D99A) yield secondary KIEs similar to that of WT, indicating that mutations at the hydrogen-bonding residues do not significantly decrease the contribution of CM/HT to the KSI reaction.

Effect of membrane lipid environment on the activity of bovine adrenal 3-oxo-Δ5-steroid isomerase

The 3-oxo-delta 5-steroid isomerase (EC 5.3.3.1) activity from bovine adrenal cortex microsomes can be extracted in soluble form by the use of appropriate detergents, although recovery of enzyme activity is low (ca. 2%). Activity is restored upon removal of detergent and reconstitution of the enzyme into phospholipid vesicles. Both Km and Vmax of 3-oxo-delta 5-steroid isomerase of intact microsomes increase as the pH is raised from 7.5 to 9.5, with a particularly sharp increase (6- to 8-fold) above pH 8.5. The kinetic parameters of a detergent-solubilized isomerase preparation show little increase from pH 7.5 to 9.0, but isomerase reconstituted into artificial phospholipid vesicles demonstrates a 6- to 10-fold increase in both Km and Vmax over this pH range. Addition of Ca++ (1 mM) enhances the pH dependence of both Km and Vmax of the membrane-bound isomerase, causing a slight rise in Vmax/Km.

Decarboxylations of β-Keto Acids and Related Compounds

The transfer of a proton from one atom to another is one of the most fundamental processes in chemistry and biochemistry. The detailed mechanism of this reaction has been given much attention both for proton transfers to and from carbon and the more electronegative oxygen and nitrogen.(1) An important class of proton transfers involves those reactions in which another bond is made or broken in addition to the proton transfer itself. Two extreme conditions may occur. The two steps of the reaction may be concerted (all bond breaking and making simultaneous) or stepwise (formation or cleavage of one bond leading to a true intermediate, followed by further bond making and/or breaking). We shall use the definition of a “true intermediate” proposed by Bauer,(2) that is, any species with a lifetime of greater than one molecular vibration: in other words a species with restoring forces for all of its vibrational motions.

Synthesis of 5,7- and 6,7-Dinitro-2-Tetralones

Abstract The synthesis of 5,7- and 6,7-dinitro-2-tetralones is described. Nitration of 2-tetralyl acetate gives 5,7- and 6,7-dinitro-2-tetralyl acetates in 43% and 18% yield, respectively. Cleavage of the acetyl residues, followed by Oppenauer oxidation provides the corresponding dinitro-2-tetralones.