THE KINETIC MECHANISM CATALYZED BY HOMOGENEOUS RAT-LIVER 3-ALPHA-HYDROXYSTEROID DEHYDROGENASE - EVIDENCE FOR BINARY AND TERNARY DEAD-END COMPLEXES CONTAINING NONSTEROIDAL ANTIINFLAMMATORY DRUGS

Rat liver 3-alpha-hydroxysteroid dehydrogenase (3-alpha-HSD) (EC 1.1.1.50) is an NAD(P)+-dependent oxidoreductase that is potently inhibited at its active site by non-steroidal anti-inflammatory drugs (NSAIDs). Initial-velocity and product-inhibition studies performed in either direction at pH 7.0 are consistent with a sequential ordered Bi Bi mechanism in which pyridine nucleotide binds first and leaves last. This mechanism is supported by fluorescence titrations of the E.NADH complex, and by the failure to detect the binding of either [H-3]androsterone or [H-3]androstanedione to free enzyme by equilibrium dialysis. Dead-end inhibition studies with NSAIDs also support this mechanism. Initial-velocity studies with indomethacin show that this drug is an uncompetitive inhibitor against NAD+, but a potent competitive inhibitor against androsterone, indicating the ordered formation of an E.NAD+.indomethacin complex. Calculation of the individual rate constants reveals that the binding and release of pyridine nucleotide is rate-limiting and that isomerization of the central complex is favoured in the forward direction. Equilibrium dialysis experiments with [C-14]indomethacin reveal the presence of two abortive NSAID complexes, a high-affinity ternary complex corresponding to E.NAD+.indomethacin (K(d) = 1-2-mu-M for indomethacin) and a low-affinity binary complex corresponding to E.indomethacin (K(d) = 22-mu-M for indomethacin). Since indomethacin has a low affinity for free enzyme, the formation of this abortive binary complex does not complicate kinetic measurements which are made in the presence of NAD+, but may contribute to the inhibition of the enzyme by NSAIDs. Using either pro-R-[4-H-3]NADH or pro-S-[4-H-3]NADH as cofactor, radiolabelled androsterone was formed only when the pro-R-[4-H-3]NADH was used, confirming that purified 3-alpha-HSD is a Class A dehydrogenase.