SmgGDS stabilizes nucleotide-bound and -free forms of the Rac1 GTP-binding protein and stimulates GTP/GDP exchange through a substituted enzyme mechanism

T. H. Chuang, X. Xu, Lawrence Quilliam, G. M. Bokoch

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

The Rac proteins, Rac1 and Rac2, are essential components of the NADPH oxidase system of phagocytes and regulate the actin assembly associated with membrane ruffling. These functions are controlled by the GTP-bound form of Rac. The biochemical interaction between Rac and its only known GDP-dissociation stimulator (termed smgGDS) was characterized. SmgGDS was able to stimulate the incorporation of guanosine 5'-[γ-thio]triphosphate GTP[γS] into the RhoA, Rac2, Rac1, Rap1A and CDC42Hs GTP-binding proteins, but the activity was greatest toward RhoA and Rac2. Isoprenoid modification of these proteins was not absolutely required for the interaction with smgGDS. Interestingly, the activity of smgGDS toward Rac1 could not be observed in a [3H]GDP/GTP exchange assay under conditions where it stimulated incorporation of GTP[γS] into Rac1. We determined that smgGDS prevented the loss of Rac1 activity during the [3H]GDP/GTP exchange assay by demonstrating the ability of smgGDS to inhibit the loss of Rac1 GTP[γS]-binding during incubations at 30°C. This stabilizing effect was exactly counterbalanced by the ability of smgGDS to stimulate the release of [3H]GDP from Rac1, thereby producing no net observable effect in the exchange assay. SmgGDS was able to effectively stimulate the release of GDP but not GTP[γS] from Rac1. SmgGDS maintains Rac1 in a nucleotide-free form after release of GDP, indicating that the reaction between Rac1 and smgGDS involves a substituted enzyme mechanism.

Original languageEnglish (US)
Pages (from-to)761-767
Number of pages7
JournalBiochemical Journal
Volume303
Issue number3
StatePublished - 1994
Externally publishedYes

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rac1 GTP-Binding Protein
Guanosine Triphosphate
Nucleotides
Enzymes
Assays
rap1 GTP-Binding Proteins
rac GTP-Binding Proteins
Guanine Nucleotide Exchange Factors
Guanosine
NADPH Oxidase
Terpenes
Phagocytes
Actins
Membranes

ASJC Scopus subject areas

  • Biochemistry

Cite this

SmgGDS stabilizes nucleotide-bound and -free forms of the Rac1 GTP-binding protein and stimulates GTP/GDP exchange through a substituted enzyme mechanism. / Chuang, T. H.; Xu, X.; Quilliam, Lawrence; Bokoch, G. M.

In: Biochemical Journal, Vol. 303, No. 3, 1994, p. 761-767.

Research output: Contribution to journalArticle

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abstract = "The Rac proteins, Rac1 and Rac2, are essential components of the NADPH oxidase system of phagocytes and regulate the actin assembly associated with membrane ruffling. These functions are controlled by the GTP-bound form of Rac. The biochemical interaction between Rac and its only known GDP-dissociation stimulator (termed smgGDS) was characterized. SmgGDS was able to stimulate the incorporation of guanosine 5'-[γ-thio]triphosphate GTP[γS] into the RhoA, Rac2, Rac1, Rap1A and CDC42Hs GTP-binding proteins, but the activity was greatest toward RhoA and Rac2. Isoprenoid modification of these proteins was not absolutely required for the interaction with smgGDS. Interestingly, the activity of smgGDS toward Rac1 could not be observed in a [3H]GDP/GTP exchange assay under conditions where it stimulated incorporation of GTP[γS] into Rac1. We determined that smgGDS prevented the loss of Rac1 activity during the [3H]GDP/GTP exchange assay by demonstrating the ability of smgGDS to inhibit the loss of Rac1 GTP[γS]-binding during incubations at 30°C. This stabilizing effect was exactly counterbalanced by the ability of smgGDS to stimulate the release of [3H]GDP from Rac1, thereby producing no net observable effect in the exchange assay. SmgGDS was able to effectively stimulate the release of GDP but not GTP[γS] from Rac1. SmgGDS maintains Rac1 in a nucleotide-free form after release of GDP, indicating that the reaction between Rac1 and smgGDS involves a substituted enzyme mechanism.",
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T1 - SmgGDS stabilizes nucleotide-bound and -free forms of the Rac1 GTP-binding protein and stimulates GTP/GDP exchange through a substituted enzyme mechanism

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AU - Bokoch, G. M.

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