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Analysis of the Kinetics of ASAP1 GAP activity
Author(s) -
Luo Ruibai,
Ahvazi Bijan,
Amariei Diana,
Shroder Deborah,
Burrola Beatriz,
Losert Wolfgang,
Randazzo Paul
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a616-b
Subject(s) - enzyme kinetics , gtp' , kinetics , gtpase , chemistry , biochemistry , arginine , biology , amino acid , enzyme , active site , physics , quantum mechanics
Arf GTPase‐activating proteins (GAPs) are enzymes that catalyze the hydrolysis of GTP bound to the small GTP‐binding protein Arf. They have also been proposed to function as Arf effectors. To understand the relationship of effector and GAP activities, we have set out to characterize the kinetics of the GAP‐induced GTP hydrolysis using a truncated form of ASAP1 as a model. To determine the Km and kcat of ASAP1, we conducted steady state and single turnover kinetics analyses, while steady kinetics were performed using FluorMax3 spectrophotometer and single turnover kinetics were carried out using a RQF‐3 rapid chemical quench‐flow. ASAP1 used Arf1·GTP as a substrate with a kcat of 66 ± 6/sec and a Km of 1.3 ± 0.4 mM determined by steady state kinetics and a kcat of 53 ± 7/sec determined by single turnover kinetics. Tetrafluoroaluminate (AlF4‐), which stabilizes complexes of other Ras family members with their cognate GAPs, also stabilized a complex of Arf1·GDP with ASAP1. Mutation of arginine 497 to a lysine affected kcat by 4 orders of magnitude. ASAP1 with arginine 497 changed to alanine or glutamine had no detectable GAP activity and an affinity for Arf1·GTP » 0.1 that of the Km of wild type protein. Changing tryptophan‐479, isoleucine‐490, arginine‐505, leucine‐511 or aspartate‐512 was predicted, based on previous studies, to affect affinity for Arf1·GTP. Instead, these mutations primarily affected the catalytic constant. In NIH 3T3 fibroblasts, [R497K]ASAP1, R505A]ASAP1 and [D512A]ASAP1 associated with actin rich dorsal ruffles, colocalizing with Arf1 in these structures, particularly, [R497K]ASAP1 can increase the dorsal ruffles comparing with the control. These results are consistent with Arf GAP ASAP1 functioning in binary complex with Arf1·GTP to induce a transition state towards GTP hydrolysis. The results have also led us to speculate that Arf1·GTP·ASAP1 can exist in at least two different conformations that are differentially stabilized by mutations.