Welsh K M, Trach K A, Folger C, Hoch J A
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.
J Bacteriol. 1994 Dec;176(23):7161-8. doi: 10.1128/jb.176.23.7161-7168.1994.
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 Km of 5.4 microM and a kcat of 0.0061 min-1 at 37 degrees C. GDP was a competitive inhibitor of this hydrolysis, with an inhibition constant of 1.7 microM at 37 degrees C. The dissociation constant for GDP from the Obg protein was 0.5 microM at 4 degrees C and was estimated to be 1.3 microM 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.
枯草芽孢杆菌的一种重要鸟嘌呤核苷酸结合蛋白Obg，已对其GTP酶活性进行了表征。该蛋白在37℃下能水解GTP，Km为5.4微摩尔，kcat为0.0061分钟-1。GDP是这种水解反应的竞争性抑制剂，在37℃下抑制常数为1.7微摩尔。在4℃时，GDP与Obg蛋白的解离常数为0.5微摩尔，在37℃时估计为1.3微摩尔。约80%的纯化蛋白能够结合GDP。除了水解GTP外，Obg还能与该底物进行自身磷酸化。随后共价磷酸的释放速率过慢，无法解释GTP水解的总体速率，这表明体外水解不是通过观察到的磷酰胺中间体进行的。据推测，该酶的磷酸化形式可能代表一种开启或关闭的构型，其中任何一种构型通常都可能由效应分子诱导。来自Obg温度敏感突变体的这种酶在非允许温度下未显示出明显改变的GTP酶活性。
