ROLES OF G(O-ALPHA) TRYPTOPHANS IN GTP HYDROLYSIS, GDP RELEASE, AND FLUORESCENCE SIGNALS

Single tryptophan mutants of a histidine-tagged G(o alpha) (W132F and W212F) were prepared to examine the functional and spectroscopic role of tryptophan in G(o alpha). The mutants bound GTP gamma S with high a ffinity and showed only modest changes in GDP affinity. GTP gamma S-st imulated intrinsic fluorescence changes were completely abolished by r emoval of W-212 but were not affected by elimination of W-132, In cont rast, both W-132 and W-212 contributed to the fluorescence signal from binding of methylanthraniloyl-GTP gamma S (mGTP gamma S). W132F and W 212F mutants showed 57% and 34% of the mGTP gamma S fluorescence chang e of wild type (WT), respectively, The decreased fluorescence signals were not due to reduced activation of the W212F protein by nucleotide as protection from tryptic digestion was unchanged. The kinetics of nu cleotide binding and hydrolysis were also altered in both mutants. GDP dissociation was slower (0.14 min(-1)) for W132F and faster (0.54 min (-1)) for W212F than for WT (0.25 min(-1)), As expected, the steady-st ate V-max for GTPase was lower for W132F, but surprisingly it was also lower for W212F despite faster GDP release, Single turnover kinetics revealed a lower k(cat) for W212F (0.52 min(-1)) compared to WT (1.39 min(-1)) and W132F (1.0 min(-1)). Thus, W212 in G(o alpha) makes a dom inant contribution to both intrinsic and extrinsic fluorescence signal s upon alpha subunit activation, In addition, both tryptophans modulat e the kinetics of nucleotide binding and hydrolysis.