The Mechanism of Water-Proton Relaxation in Enzyme•Paramagnetic-Ion Complexes.

The water proton-relaxation times, T₁ and T₂have been measured in solutions of Mn(II) and ATP in the presence and absence of rabbit muscle phosphofructokinase over a wide range of temperatures and nuclear frequencies, using pulsed nuclear magnetic resonance techniques. The equilibrium between the enzyme and the Mn · ATP complex has also been studied as a function of temperature, and the relaxation rates in the bound complex determined. An analysis of the relaxation rates in both free and enzyme-bound Mn · ATP sites according to established theories of paramagnetic relaxation has been carried out by means of graphical and computing methods, and yielded estimates of the Mn(II) water co-ordination number (q) and the relevant correlation times their frequency and temperature dependence. Relaxation in the ternary enzyme complex is dipolar in nature and the correlation time (Τ) is dominated by the water residence time (Τ_M) or the Mn electron spin relaxation time (Τ_s) according to frequency. The values obtained for both Τ_c and &Tau_s are longer than earlier estimates. In the binary Mn · ATP complex the dipolar interactions are dominated by rotation (Τ_R) but in addition there is a large scalar interaction governed by both Τ_M and Τ_s. The significance of the parameters in the structure if the ternary enzyme complex is discussed, together with the implications of the revised parameters &Tau_c and &Tau_s for earlier mapping studies of the Mn · ATP sites and the spin-labelled enzyme. The limitations of the analysis have been stressed throughout. [ABSTRACT FROM AUTHOR]