MAGNETIC-RESONANCE AND KINETIC STUDIES OF THE MANGANESE(II) ION AND SUBSTRATE COMPLEXES OF THE CATALYTIC SUBUNIT OF ADENOSINE-3',5'-MONOPHOSPHATE DEPENDENT PROTEIN-KINASE FROM BOVINE HEART

The catalytic subunit of cAMP-dependent protein kinase from bovine heart binds Mn2+ very weakly (KD ≥ 1 mM) as determined by electron paramagnetic resonance and by the enhancement of the paramagnetic effect of Mn2+ on the longitudinal relaxation rate 1/T1p of water protons. The presence of a nucleotide such as ADP or AMP-PCP causes the appearance of two tight Mn2+ binding sites per molecule of enzyme which enhance the effects of Mn2+ on 1/T1p of water protons by factors (ε) of 4 to 7. Titration of the enzyme with ADP in the presence of excess Mn2+, monitoring the increase in the enhancement factor, reveals 0.95 tight ADP binding sites per enzyme molecule. In the enzyme-ADP complex, the affinities of Mn2+ for the two binding sites are equal (KD=19 μM), while in the ternary complex of enzyme, ADP, and the heptapeptide substrate, Leu-Arg-Arg-AlaSer-Leu-Gly, and in the binary enzyme-AMP-PCP complex the affinities for Mn2+ at the two sites are unequal (KD1=6-10 μM; KD2=50-60 μM). The presence of the substitution-inert complex Co3+(NH3)4ATP causes the appearance of only one Mn2+ binding site on the enzyme (KD=130 -μM, ε=9.6), indicating that the tighter of the two Mn2+ binding sites in the E-ADP-peptide and E-AMP-PCP complexes is on the enzyme-bound nucleotide while the more weakly bound Mn2+ is partially or entirely on the protein. A kinetic study of the enzyme-catalyzed phosphorylation of the peptide substrate reveals both activation and 50-fold inhibition by Mn2+. Analysis of the data assuming a rapid equilibrium kinetic scheme yields an activator constant (3 μM) and an inhibitor constant (29 μM) for Mn2+ comparable to the dissociation constants of Mn2+ from the nucleotide site (6-10 μM) and protein site (50-60 μM), respectively, as determined in the binding studies. These findings indicate that the nucleotide-bound Mn2+ activates, while the more weakly bound Mn2+ is inhibitory, due to direct or indirect effects on the active site. Analogous results, differing only quantitatively, are obtained with Mg2+. Thus, by studies of the binding of Mg2+ in competition with Mn2+, two Mg2+ binding sites (KD=1.6 mM) are detected on the E-ADP complex, one of which activates and the other of which inhibits fivefold, as suggested by kinetic studies. Interactions between the inhibitory Mn2+ site and the active nucleotide-Mn2+ site on the enzyme are detected as a mutual tightening of binding and as an increase in enhancement at the inhibitory site when the active site binds a metal-nucleotide complex. The kinetically determined Km values of ATP (69 μM) and of Mn2+-ATP (16 μM) are in good agreement with the dissociation constants of nucleotides (65-78 μM) and Mn2+-nucleotides (21-30 μM), respectively, as determined in the binding studies. This agreement indicates active site binding of the nucleotides and supports the validity of the rapid equilibrium kinetic scheme for the Mn2+-activated enzyme. The requirement for nucleotides for the tight binding of Mn2+ to the enzyme and the ability of the substitution-inert species Co3+(NH3)4ATP to bind to the nucleotide site suggest that the coordination scheme of the active ternary complex is a nucleotide bridge or enzyme-ATP-metal complex. © 1979, American Chemical Society. All rights reserved.