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

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‐Mg 2+ concetrations. Under those conditions, (45 nm free Mg 2+ ), 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 Mg 2+ was mainly due to an increased K cat , 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 ( K off·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 ( k off · 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 (C 20 ) but not for farnesylation (C 15 , 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 k off · GDP of [Thr115 → Asn]rac1 was reduced to that of H‐ras, but [Thr115 → Asn]rac1 exhibited essentially identical k cat (0.13 min −1 at 20°C) and k off · 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 k cat of rac1 is not within the consensus amino acids of the four guanine‐nucleotide‐binding domains.