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-Mg2+ concentrations. 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 almost-equal-to 0.39 min-1 at 37-degrees-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 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-degrees-C). Rac1 also released bound GDP faster than H-ras (k(off.GDP) = 1.02 min-1 for rac1 vs 0.33 min-1 for H-ras at 20-degrees-C). In contrast, rac1 released bound guanosine 5'-[gamma-thioltriphosphate (GTP[S]) at a slower rate than H-ras (k(off. GTP[S]) almost-equal-to 0.04 min-1 for rac1 vs 0.31 min-1 for H-ras at 20-degrees-C). Rac1 was a very good substrate for in vitro geranylgeranylation (C20) 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-degrees-C) and k(off.GTP[S]) (0.03 min-1 at 20-degrees-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.