Magnetic resonance measurements of intersubstrate distances at the active site of protein kinase using substitution-inert cobalt(III) and chromium(III) complexes of adenosine 5'-(.beta.,.gamma.-methylenetriphosphate)
Co3+ and Cr3+ complexes of .beta.,.gamma.-methylene-ATP (AMPPCP), which are substitution-inert substrate analogs inactive in phosphoryl transfer reactions, were used in binding and structural studies of [bovine heart muscle] 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 EPR 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 .ANG. in the absence of enzyme and 5.0 .+-. 0.9 .ANG. 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 to calculate the distance between the 2 metal ions of 4.8 .+-. 0.4 .ANG.. 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. 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 .ANG., respectively, were calculated. These and previous measurements were used to estimate a distance of 5.3 .+-. 0.7 and along the reaction coordinate between the .gamma.-phosphorus of ATP and the serine hydroxyl O2. This distance is 2 .ANG. greater than that required for molecular contact. The mechanistic implications of these findings are discussed.