Rac1, a low‐molecular‐mass GTP‐binding‐protein with high intrinsic GTPase activity and distinct biochemical properties

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Abstract
Rac1, a member of the family of low-molecular-mass GTP-binding proteins, functions in phagocytic leukocytes as a component necessary for activation of the respiratory burst. To characterize the biochemical properties of rac1, the protein was expressed as a fusion protein in Escherichia coli and purified to greater than 99% homogeneity by affinity chromatography. Rac1 protein bound maximally bound and hydrolyzed GTP under low free-Mg2+ concetrations. Under those conditions, (45 nm free Mg2+), purified rac1 exhibited a steady-state GTPase activity of 18 nmol · min−1· mg protein−1 (turnover number ∼ 0.39 min−1 at 37°C), which is 40-fold higher than H-ras. The high intrinsic GTPase activity of rac1 under low free Mg2+ was mainly due to an increased Kcat, the rate constant for hydrolysis of bound GTP, which was 0.29 min−1 for rac1 vs 0.007 min−1 for H-ras (at 20°C). Rac1 also released bound GDP faster than H-ras (Koff·sGDP= 1.02 min−1 for rac1 vs0.33 min−1 for H-ras at 20°C). In contrast, rac1 released bound guanosine 5′-[γ-thio]triphosphate (GTP[S] at a slower rate than H-ras (koff · GTP[S]∼ 0.04 min−1 for rac1 vs 0.31 min−1 for H-ras at 20°C). Rac1 was a very good substrate for in vitro geranylgernylation (C20) but not for farnesylation (C15, whereas the converse is true for H-ras. Surprisingly, rac1 was a very poor substrate for in vitro ADP-ribosylation by the C3 component of Clostridium botulinum toxin compared to rhoA. As a further characterization of rac1, a mutant was made in which the Thr115 was replaced by asparagine. This protein (referred to as [Thr115 → Asn]rac1) contains the consensus amino acids of all four GTP-binding domains of H-ras. The koff·GDP of [Thr115 → Asn]rac1 was reduced to that of H-ras, but [Thr115 → Asn]rac1 exhibited essentially identical kcat (0.13 min−1 at 20°C) and koff·GTP[s] (0.03 min−1 at 20°C) values as the Wild-type protein. thus, the region(s) in rac1 which control the dissociation of GTP[S] (and presumably GTP) do not entirely coincide with those controlling GDP dissociation. Biochemical analysis of [Thr115 → Asn]rac1 also suggests that the region responsible for the increased kcat of rac1 is not within the consensus amino acids of the four guanine-nucleotide-binding domains.