JBC, Vol. 252, Issue 1, 205-211, Jan, 1977
20beta-Hydroxysteroid oxidoreductase. Kinetics and binding of corticosteroids and corticosteroid-21-aldehydes
E. S. Szymanski and C. S. Furfine
Corticosteroid-21-aldehydes were reduced only at C-20 by 20
beta-hydroxysteroid dehydrogenase (EC 1.1.1.53) of Streptomyces
hydrogenans, and the reduction occurred by transfer of hydrogen from the
B-side of NADH. A kinetic investigation of cortisol, cortisone,
cortexolone, and the 21-aldehydes of each indicated: (a) the magnitude of
the Michaelis constant for any substrate was independent of the second
substrate concentration; (b) the 21-aldehydes had larger Michaelis
constants (5- to 8-fold) and larger maximum velocities (16- to 40-fold)
than the steroids from which they were synthesized; (c) the Michaelis
constant for NADH, 29 muM, was independent of the steroid substrate. With
cortisol and cortisol-21-aldehyde, product inhibition patterns showed only
slope effects with steroid product and NAD+, suggesting a "random"
mechanism. Inhibition studies with the "poor" substrate cortisol indicated
that cortisol and cortisol-21-aldehyde were reduced at the same site. The
inhibition constant (180 muM) agreed with the Michaelis constant of
cortisol (140 muM). The steroid product, 20beta-hydroxyprogesterone, gives
noncompetitive inhibition patterns with respect to NADH and
cortisol-21-aldehyde, indicating a separate binding site exists on the
enzyme for this inhibitor. The intrinsic protein fluorescence of
20beta-hydroxysteroid dehydrogenase was quenched by NADH (56%) with a
dissociation constant of 16 muM. NAD" quenched the protein fluorescence
somewhat less (31%) with a dissociation constant of 104 muM. The
fluorescence of 2-p-toluidine-6-naphthalene sulfonate is enhanced in the
presence of enzyme, and there is a blue shift in the emission wavelength
maximum. The enzyme-enhanced 2-p-toluidine-6-naphthalene sulfonate
fluorescence is quenched by NAD+ (32%) with a dissociation constant of 128
muM. Corticosteroids and their corresponding 21-aldehydes completely quench
the enhanced 2-p-toluidine-6-naphthalene sulfonate fluorescence and this
feature can be used to determine enzyme-steroid dissociation constants.
Corticosteroid-21-aldehydes and NAD+ dissociation constants determined in
this manner agree with values obtained in kinetic measurements. The
dissociation constants determined for cortisol, cortisone, cortexolone,
progesterone, and 20beta-hydroxyprogesterone were at least 1 order of
magnitude greater than the corresponding kinetic constants, and these
findings suggest the presence of a kinetically insignificant binding site.
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