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Question: Small GTPases are generally active in the GTP-bound state and inactive when the GTPis hydrolyzed …


Small GTPases are generally active in the GTP-bound state and inactive when the GTPis hydrolyzed to GDP. In the absence of a GTPase activating protein (GAP), small GTPases typically hydrolyze GTP very slowly. The mechanism by which GAP stimulates GTP hydrolysis is known for the small GTPase Ras. When Ras-GAP binds to Ras, it alters the conformation of Ras and provides a critical, catalytic arginine finger that stabilizes the transition state for GTP hydrolysis, thereby stimulating hydrolysis by several orders of magnitude. During assembly of COPI-coated vesicles, ARF1, a small GTPase, binds to ARF1-GAP, which locks ARF1 into its active catalytic conformation, but does not supply the catalytic arginine. Since COPI subunits also bind to ARF1, you wonder if they might affect GTP hydrolysis. To test this possibility, you mix ARF1, ARF1-GAP, and COPI subunits in various combinations and measure GTP hydrolysis (see Table). TABLE 13-1 Rates of GTP hydrolysis by various combinations of ARF1, ARF1-GAP, and COPI subunits (Problem 13-25). Components added ARF1 ARF1 ARF1-GAP ARF1 +COPI subunits ARF1 ARF1-GAPCOPI subunits Rate of GTP hydrolysis 0 0 1000

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Small GTPases are generally active in the GTP-bound state and inactive when the GTPis hydrolyzed to GDP. In the absence of a GTPase activating protein (GAP), small GTPases typically hydrolyze GTP very slowly. The mechanism by which GAP stimulates GTP hydrolysis is known for the small GTPase Ras. When Ras-GAP binds to Ras, it alters the conformation of Ras and provides a critical, catalytic arginine "finger" that stabilizes the transition state for GTP hydrolysis, thereby stimulating hydrolysis by several orders of magnitude. During assembly of COPI-coated vesicles, ARF1, a small GTPase, binds to ARF1-GAP, which locks ARF1 into its active catalytic conformation, but does not supply the catalytic arginine. Since COPI subunits also bind to ARF1, you wonder if they might affect GTP hydrolysis. To test this possibility, you mix ARF1, ARF1-GAP, and COPI subunits in various combinations and measure GTP hydrolysis (see Table). TABLE 13-1 Rates of GTP hydrolysis by various combinations of ARF1, ARF1-GAP, and COPI subunits (Problem 13-25). Components added ARF1 ARF1 ARF1-GAP ARF1 +COPI subunits ARF1 ARF1-GAPCOPI subunits Rate of GTP hydrolysis 0 0 1000

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