Proton nuclear magnetic resonance studies of Pseudomonas testosteroni 3-oxo- Delta super(5)-steroid isomerase and its interaction with 17 beta -estradiol

3-Oxo- Delta super(5)-steroid isomerase (EC 5.3.3.1) and its complex with the competitive inhibitor 17 beta -estradiol have been studied by super(1)H NMR at 360 MHz. The histidine C-2 protons of the enzyme give rise to distinct peaks in the spectral region below 7.3 ppm. In the absence of 17 beta -estradiol the pK sub(a)' values obtained were 4.68, 5.94, and 7.71, while in the presence of 17 beta -estradiol the pK sub(a)' values of the histidines were 4.90, 5.83, and 7.61. Binding of 17 beta -estradiol results in substantial changes in the aromatic region of the spectrum. The resonances of 4.8-7.8 protein aromatic protons undergo an upfield shift as a result of 17 beta -estradiol binding; however, the structural basis for this perturbation is not yet known. The spectrum of the enzyme contains several resonances which are unusually narrow for a molecule of molecular weight 27,000. Several types of evidence indicate that these resonances arise from groups of the protein rather than from contaminating small molecules.