BIOCHEMICAL-CHARACTERIZATION OF THE ESSENTIAL GTP-BINDING PROTEIN OBG OF BACILLUS-SUBTILIS

An essential guanine nucleotide-binding protein, Obg, of Bacillus subtilis has been characterized with respect to its enzymatic activity for GTP. The protein was seen to hydrolyze GTP with a K-m of 5.4 mu M and a k(cat) of 0.0061 min(-1) at 37 degrees C. GDP was a competitive inhibitor of this hydrolysis, with an inhibition constant of 1.7 mu M at 37 degrees C. The dissociation constant for GDP from the Obg protein was 0.5 mu M at 4 degrees C and was estimated to be 1.3 mu M at 37 degrees C. Approximately 80% of the purified protein was capable of binding GDP. In addition to hydrolysis of GTP, Obg was seen to autophosphorylate with this substrate. Subsequent release of the covalent phosphate proceeds at too slow a rate to account for the overall rate of GTP hydrolysis, indicating that in vitro hydrolysis does not proceed via the observed phosphoamidate intermediate. It was speculated that the phosphorylated form of the enzyme may represent either a switched-on or a switched-off configuration, either of which may be normally induced by an effector molecule. This enzyme from a temperature-sensitive mutant of Obg did not show significantly altered GTPase activity at the nonpermissive temperature.