Mechanistic studies with solubilized rat liver steroid 5 alpha-reductase: elucidation of the kinetic mechanism.
A solubilized preparation of steroid 5 alpha-reductase (EC 1.3.1.30) from rat liver has been used in studies focused toward an understanding of the kinetic mechanism associated with enzyme catalysis. From the results of analyses with product and dead-end inhibitors, a preferentially ordered binding of substrates and release of products from the surface of the enzyme is proposed. The observations from these experiments were identical with those using the steroid 5 alpha-reductase activity associated with rat liver microsomes. The primary isotope effects on steady-state kinetic parameters when [4S-2H]NADPH was used also were consistent with an ordered kinetic mechanism. Normal isotope effects were observed for all three kinetic parameters (Vm/Km for both testosterone and NADPH and Vm) at all substrate concentrations used experimentally. Upon extrapolation to infinite concentration of testosterone, the isotope effect on Vm/Km for NADPH approached unity, indicating that the nicotinamide dinucleotide phosphate is the first substrate binding to and the second product released from the enzyme. The isotope effects on Vm/Km for testosterone at infinite concentration of cofactor and on Vm were 3.8 +/- 0.5 and 3.3 +/- 0.4, respectively. Data from the pH profiles of these three steady-state parameters and the inhibition constants (1/Ki) of competitive inhibitors versus both substrates indicate that the binding of nicotinamide dinucleotide phosphate involves coordination of its anionic 2'-phosphate to a protonated enzyme-associated base with an apparent pK near 8.0. From these results, relative limits have been placed on several of the internal rate constants used to describe the ordered mechanism of the rat liver steroid 5 alpha-reductase.
Levy
M A
MA
Department of Medicinal Chemistry, Smith Kline & French Laboratories, King of Prussia, Pennsylvania 19406.
Brandt
M
M
Greway
A T
AT
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000378:metabolism*; D058891:5-alpha Reductase Inhibitors; D000818:Animals; D003903:Deuterium; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009536:Niacinamide / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility
D058891:5-alpha Reductase Inhibitors; D009536:Niacinamide; D003903:Deuterium; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase