期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2010
卷号：107
期号：21
页码：9596-9601
DOI：10.1073/pnas.1004803107
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：When G-protein [α] subunits binds GTP and Mg2+, they transition from their inactive to their active conformation. This transition is accompanied by completion of the coordination shell of Mg2+ with electrons from six oxygens: two water molecules, the {beta} and {gamma} phosphoryls of GTP, a helix-[α]1 Ser, and a switch I domain (SWI) Thr, and the repositioning of SWI and SWII domains. SWII binds and regulates effector enzymes and facilitates GTP hydrolysis by repositioning the {gamma}-carbonyl of a Gln. Mutating the Ser generates regulatory GTPases that cannot lock Mg2+ into its place and are locked in their inactive state with dominant negative properties. Curiously, mutating the Thr appears to reduce GTP hydrolysis. The reason for this difference is not known because it is also not known why removal of the Thr should affect the overall GTPase cycle differently than removal of the Ser. Working with recombinant Gs[α], we report that mutating its SWI-Thr to either Ala, Glu, Gln, or Asp results not only in diminished GTPase activity but also in spontaneous activation of the SWII domain. Upon close examination of existing [α] subunit crystals, we noted the oxygen of the backbone carbonyl of SWI-Thr and of the {gamma}-carbonyl of SWII Gln to be roughly equidistant from the oxygen of the hydrolytic H2O. Our observations indicate that the Gln and Thr carbonyls play equihierarchical roles in the GTPase process and provide the mechanism that explains why mutating the Thr mimics mutating the Gln and not that of the Ser.
关键词：ensymes/hydrolases ; protein/metal ion interaction ; catalytic water ; heterotrimeric G proteins
