Roles of Go Tryptophans in GTP Hydrolysis, GDP Release, and Fluorescence Signals
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- 作者：Keng-Li Lan ; Ann E. Remmers ; and Richard R. Neubig
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：January 20, 1998
- 年：1998
- 卷：37
- 期：3
- 页码：837 - 843
- 全文大小：90K
- 年卷期：v.37,no.3(January 20, 1998)
- ISSN：1520-4995
文摘
Single tryptophan mutants of a histidine-taggedGo
(W132F and W212F) were preparedtoexamine the functional and spectroscopic role of tryptophan inGo. The mutants bound GTP
Swithhigh affinity and showed only modest changes in GDP affinity.GTPS-stimulated intrinsic fluorescencechanges were completely abolished by removal of W212 butwere not affected by elimination of W132.Incontrast, both W132 and W212 contributed to thefluorescence signal from binding of methylanthraniloyl-GTPS (mGTPS). W132F and W212F mutants showed 57% and 34% ofthe mGTPS fluorescencechange of wild type (WT), respectively. The decreased fluorescencesignals were not due to reducedactivation of the W212F protein by nucleotide as protection fromtryptic digestion was unchanged. Thekinetics of nucleotide binding and hydrolysis were also altered in bothmutants. GDP dissociation wasslower (0.14 min-1) for W132F and faster(0.54 min-1) for W212F than for WT (0.25min-1). As expected,the steady-state Vmax for GTPase was lower forW132F, but surprisingly it was also lower for W212Fdespite faster GDP release. Single turnover kinetics revealed alower kcat for W212F (0.52min-1) comparedto WT (1.39 min-1) and W132F (1.0min-1). Thus, W212 inGo makes a dominant contribution tobothintrinsic and extrinsic fluorescence signals upon subunitactivation. In addition, both tryptophansmodulate the kinetics of nucleotide binding andhydrolysis.
(W132F and W212F) were preparedtoexamine the functional and spectroscopic role of tryptophan inGo. The mutants bound GTPSwithhigh affinity and showed only modest changes in GDP affinity.GTPS-stimulated intrinsic fluorescencechanges were completely abolished by removal of W212 butwere not affected by elimination of W132.Incontrast, both W132 and W212 contributed to thefluorescence signal from binding of methylanthraniloyl-GTPS (mGTPS). W132F and W212F mutants showed 57% and 34% ofthe mGTPS fluorescencechange of wild type (WT), respectively. The decreased fluorescencesignals were not due to reducedactivation of the W212F protein by nucleotide as protection fromtryptic digestion was unchanged. Thekinetics of nucleotide binding and hydrolysis were also altered in bothmutants. GDP dissociation wasslower (0.14 min-1) for W132F and faster(0.54 min-1) for W212F than for WT (0.25min-1). As expected,the steady-state Vmax for GTPase was lower forW132F, but surprisingly it was also lower for W212Fdespite faster GDP release. Single turnover kinetics revealed alower kcat for W212F (0.52min-1) comparedto WT (1.39 min-1) and W132F (1.0min-1). Thus, W212 inGo makes a dominant contribution tobothintrinsic and extrinsic fluorescence signals upon subunitactivation. In addition, both tryptophansmodulate the kinetics of nucleotide binding andhydrolysis.