Multiparameter study of denaturation of 20 beta-hydroxysteroid dehydrogenase by urea in the presence of stabilizing agents.
We investigated the denaturation of tetrameric 20 beta-hydroxysteroid dehydrogenase (20R)-17 beta,20 beta,21-trihydroxysteroid:NAD+ oxidoreductase, EC 1.1.1.53) to find out whether intermediate states are formed during the process. The denaturation process was studied in the presence and absence of stabilizers, both specific, such as NADH, and non-specific, such as the salting-out anion phosphate. Changes in enzymatic activity, intrinsic protein fluorescence and far-ultraviolet circular dichroism were monitored. When NADH was present, denaturation of the enzyme by urea was a one-step transition between the native and the completely denatured state. In dilute phosphate, and even more so in concentrated phosphate, the existence of intermediate states with different stability is evidenced by the noncoincidence of the transition curves that probe for different functional and conformational aspects of the enzyme. Therefore, for 20 beta-hydroxysteroid dehydrogenase the formation of intermediates can be prevented by adding NADH, or enhanced by adding concentrated phosphate.
D015089:20-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors*; D002942:Circular Dichroism; D003349:Cortisone Reductase / Q000037:antagonists & inhibitors*; D004795:Enzyme Stability; D007700:Kinetics; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000494:pharmacology*
D014508:Urea; D015089:20-Hydroxysteroid Dehydrogenases; D003349:Cortisone Reductase