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

Rac1, a low‐molecular‐mass GTP‐binding‐protein with high intrinsic GTPase activity and distinct... 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Febs Journal Wiley

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

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References (46)

Publisher
Wiley
Copyright
Copyright © 1992 Wiley Subscription Services, Inc., A Wiley Company
ISSN
1742-464X
eISSN
1742-4658
DOI
10.1111/j.1432-1033.1992.tb16957.x
Publisher site
See Article on Publisher Site

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.

Journal

The Febs Journal – Wiley

Published: Jun 1, 1992

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