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

Eur J Biochem. 1992 Jun 1;206(2):537-46. doi: 10.1111/j.1432-1033.1992.tb16957.x.


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 approximately 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 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 degrees C). Rac1 also released bound GDP faster than H-ras (koff.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-thio]triphosphate (GTP[S]) at a slower rate than H-ras (koff.GTP[S] approximately 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 (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 degrees C) and koff-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 kcat of rac1 is not within the consensus amino acids of the four guanine-nucleotide-binding domains.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Diphosphate Ribose / metabolism
  • Amino Acid Sequence
  • Autoradiography
  • Chromatography, Gel
  • Complement C3 / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • GTP Phosphohydrolases / metabolism*
  • GTP-Binding Proteins / genetics
  • GTP-Binding Proteins / isolation & purification
  • GTP-Binding Proteins / metabolism*
  • Genes, Bacterial
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
  • Guanosine Diphosphate / metabolism
  • Hydrolysis
  • Kinetics
  • Molecular Sequence Data
  • Molecular Weight
  • Mutagenesis, Site-Directed
  • rac GTP-Binding Proteins


  • Complement C3
  • Guanosine Diphosphate
  • Adenosine Diphosphate Ribose
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • GTP Phosphohydrolases
  • GTP-Binding Proteins
  • rac GTP-Binding Proteins