Electron-paramagnetic-resonance studies of manganese(II) complexes with elongation factor Tu from Bacillus stearothermophilus. Observation of a GTP hydrolysis intermediate state complex

Abstract

Changes in the coordination of Mn²⁺ to nucleotide, water and protein at the active site of elongation factor Tu (EF-Tu) have been studied by electron paramagnetic resonance (EPR) spectroscopy. From the time dependence of the Mn²⁺ spectrum after addition of GTP to EF-Tu · Mn, it was apparent that three complexes with different EPR linewidths could be detected. Using additional information from the kinetics of ³²P_i production and release from EF-Tu · Mn · [γ-³²P]GTP these were identified as EF-Tu · Mn · GTP (linewidth 4.2 mT), EF-Tu · Mn · GDP · P_i (1.20 mT) and EF-Tu · Mn · GDP (1.29 mT). The linewidth for EF-Tu · Mn was 1.51 mT. The rate constant for GTP cleavage on EF-Tu was 0.01 min⁻¹ at 24 °C, for P_i release from the EF-Tu · GDP · P_i complex 0.0033 min⁻¹. The corresponding rate constants in the presence of Mg²⁺ were 0.003 min⁻¹ and 0.0065 min⁻¹. The rate constant for reversal of the cleavage step was found to be much smaller than that for the rate of P_i release (and consequently much smaller than in the forward direction), as shown by ³¹P-NMR experiments on the incorporation of ¹⁸O into P_i from GTP hydrolyzed in the presence of H₂¹⁸O. EPR experiments using specifically ¹⁷O-labelled GTPs demonstrated an interaction of Mn²⁺ with the β-phosphate in both the EF-Tu · GDP · P_i and EF-Tu · GDP complexes. Inorganic phosphate in the EF-Tu · GDP · P_i complex was found not to interact with the metal ion. From EPR experiments in H₂¹⁷O, it was concluded that the most probable number of water molecules in the different complexes was 4 (EF-Tu · Mn), 5 (EF-Tu · Mn · GDP · P_i) and 3 (EF-Tu · Mn · GDP), with 2, 0 and 2 metal-protein interactions respectively.