Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) are enzymes that catalyse the hydrolysis of GTP bound to the small GTP-binding protein Arf. They have also been proposed to function as Arf effectors and oncogenes. We have set out to characterize the kinetics of the GAP-induced GTP hydrolysis using a truncated form of ASAP1 [Arf GAP with SH3 (Src homology 3) domain, ankyrin repeats and PH (pleckstrin homology) domains 1] as a model. We found that ASAP1 used Arf1-GTP as a substrate with a k(cat) of 57+/-5 s(-1) and a K(m) of 2.2+/-0.5 microM determined by steady-state kinetics and a kcat of 56+/-7 s(-1) 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. As anticipated, mutation of Arg-497 to a lysine residue affected kcat to a much greater extent than K(m). Changing Trp-479, Iso-490, Arg-505, Leu-511 or Asp-512 was predicted, based on previous studies, to affect affinity for Arf1-GTP. Instead, these mutations primarily affected the k(cat). Mutants that lacked activity in vitro similarly lacked activity in an in vivo assay of ASAP1 function, the inhibition of dorsal ruffle formation. Our results support the conclusion that the Arf GAP ASAP1 functions in binary complex with Arf1-GTP to induce a transition state towards GTP hydrolysis. The results have led us to speculate that Arf1-GTP-ASAP1 undergoes a significant conformational change when transitioning from the ground to catalytically active state. The ramifications for the putative effector function of ASAP1 are discussed.

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Phenylalanine ammonia-lyases (PALs) catalyse the non-oxidative deamination of l-phenylalanine to trans-cinnamic acid, while in the presence of high ammonia concentration the reverse reaction occurs. PALs have been intensively studied, however, their industrial applications for amino acids synthesis remained limited, mainly due to their decreased operational stability or limited substrate specificity. The application of extensive directed evolution procedures to improve their stability, activity or selectivity, is hindered by the lack of reliable activity assays allowing facile screening of PAL-activity within large-sized mutant libraries. Herein, we describe the development of an enzyme-coupled fluorescent assay applicable for PAL-activity screens at whole cell level, involving decarboxylation of trans-cinnamic acid (the product of the PAL reaction) by ferulic acid decarboxylase (FDC1) and a photochemical reaction of the produced styrene with a diaryltetrazole, that generates a detectable, fluorescent pyrazoline product. The general applicability of the fluorescent assay for PALs of different origin, as well as its versatility for the detection of tyrosine ammonia-lyase (TAL) activity have been also demonstrated. Accordingly, the developed procedure provides a facile tool for the efficient activity screens of large mutant libraries of PALs in presence of non-natural substrates of interest, being essential for the substrate-specificity modifications/tailoring of PALs through directed evolution-based protein engineering.

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.