Studies of Manganous Nucleotide Complexes with Uridine Diphosphate-Glucose Pyrophosphorylase, Formyltetrahydrofolate Synthetase, and Creatine Kinase: MECHANISM OF WATER PROTON MAGNETIC RELAXATION FROM FREQUENCY DEPENDENCE MEASUREMENTS

Abstract

Abstract The longitudinal proton magnetic relaxation rate (PRR) of water in manganous complexes has been measured as a function of frequency. The ternary complexes of manganous ion, nucleotide, and enzyme for three enzymes namely, UDP-glucose pyrophosphorylase, formyl tetrahydrofolate synthetase, and creatine kinase, all exhibited a maximum in the value of the relaxation rate between 8 and 25 MHz. This observation shows that the correlation time which modulates the dipolar interaction between the electron and nuclear spins is frequency-dependent and must therefore be ascribed to the electron spin relaxation time. The enhancement of the PRR of water for the ternary complexes relative to that of the Mn(II) aquo-ion arises because the electron relaxation times for Mn(II) in the ternary complexes (10-8 to ∼10-9 sec) are much longer than the rotational correlation time (∼3 x 10-11 sec) for the simple aquo complex. The rotational modulation of the dipolar interaction for the ternary complexes is slower than modulation from electron spin relaxation and is no longer the dominant correlation time in the macromolecular complexes since their tumbling times are of the order of 10-8 to 10-7 sec. In the case of creatine kinase, the PRR of the abortive quaternary complex, E-Mn-ADP-creatine, has been deenhanced relative to the ternary complex, most markedly in the presence of nitrate ion, and the PRR increases monotonically with decreasing frequency. The number of water ligands in the first coordination sphere of Mn(II) in the quaternary complex is calculated from the relaxation rate and its frequency dependence to be less than one-half. The water proton relaxation rates obtained at various frequencies and temperatures point to the involvement of only outer sphere relaxation in the quaternary complex. It is concluded that a conformational change in the enzyme has occurred upon addition of the second substrate which makes the Mn(II) inaccessible to the solvent water. The binding of Mn(II) in the ternary MnUTP complex with UDP-glucose pyrophosphorylase is less than in the binary MnUTP complex in the absence of enzyme. The behavior of this nucleotidyl-transferring enzyme differs in this regard from phosphoryl-transferring enzymes from which it is inferred that the coordination scheme of Mn(II) may also differ.