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Co3+ and Cr3+ complexes of beta, gamma-methylene-ATP (AMPPCP), which are substitution-inert substrate analogues inactive in phosphoryl transfer reactions, have been used in binding and structural studies of cAMP-dependent protein kinase. Dissociation constants of enzyme complexes with Co(NH3)4AMPPCP and CrAMPPCP and with Mn2+, which binds at an inhibitory site, were determined by electron paramagnetic resonance and by proton relaxation rate enhancement techniques. Nuclear relaxation rate measurements at 100 and 360 MHz were used to determine the distance between Mn2+ and the beta, gamma-methylene protons of Co-(NH3)4AMPPCP, yielding 7.4 +/- 0.6 A in the absence of enzyme and 5.0 +/- 0.9 A when both Mn2+ and Co-(NH3)4AMPPCP were bound to the enzyme. The effect of the paramagnetic CrAMPPCP on the electron spin relaxation time of the enzyme-bound Mn2+ was used used to calculate the distance between the two metal ions of 4.8 +/- 0.4 A. This distance and the Mn2+-methylene distance are consistent with the previous finding that the inhibitory metal bridges the enzyme to the triphosphate chain of the enzyme-bound nucleotide [Granot, J., Kondo, H., Armstrong, R. M., Mildvan, A. S., & Kaiser, E. T. (1979) Biochemistry 18, 2339]. From the paramagnetic effects on the relaxation rates of the protons of the peptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly, distances from Mn2+ and Cr3+ to the serine methylene protons of 9.1 +/- 0.9 and 8.1 +/- 0.8 A, respectively, were calculated. These and previous measurements were used to estimate a distance of 5.3 +/- 0.7 A along the reaction coordinate between the gamma-phosphorus of ATP and the serine hydroxyl oxygen. This distance is 2 A greater than that required for molecular contact. The mechanistic implications of these findings are discussed.

17 beta-(N-tert-butylcarbamoyl)-4-aza-5 alpha-androstan-1-en-3-one (finasteride), which has been approved for treatment of benign prostatic hyperplasia, is shown here to be a slow time-dependent inhibitor of human steroid 5 alpha-reductase isozyme 1. This inhibition is characterized by an initial, fast step where the inhibitor binds to the enzyme followed by a slow step that leads to a final enzyme-inhibitor complex (EI*). No recovery of activity from this EI* complex was observed after dialysis for 3 days. The formation of EI* is diminished in the presence of a competitive, reversible inhibitor, indicating that the inhibition is active site-directed. At 37 degrees C and pH 7.0, the rate constant for the second, slow inhibition step, k3, is (1.40 +/- 0.04) x 10(-3) s-1 and the pseudo-bimolecular rate constant, k3/Ki, is (4.0 +/- 0.3) x 10(3) M-1 s-1. This latter rate constant is less than the value of 2.7 x 10(5) M-1 s-1 determined for the inhibition of 5 alpha-reductase 2 by finasteride [Faller, B., Farley, D., & Nick, H. (1993) Biochemistry 32, 5705-5710].(ABSTRACT TRUNCATED AT 250 WORDS)

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