γ-phosphate protonation and pH-dependent unfolding of the Ras·GTP·Mg2+ complex: A vibrational spectroscopy study

Hu Cheng, Sean Sukal, Robert Callender, Thomas S. Leyh

Research output: Contribution to journalArticle

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Abstract

The interdependence of GTP hydrolysis and the second messenger functions of virtually all GTPases has stimulated intensive study of the chemical mechanism of the hydrolysis. Despite numerous mutagenesis studies, the presumed general base, whose role is to activate hydrolysis by abstracting a proton from the nucleophilic water, has not been identified. Recent theoretical and experimental work suggest that the γ-phosphate of GTP could be the general base. The current study investigates this possibility by studying the pH dependence of the vibrational spectrum of the Ras·GTP·Mg 2+ and Ras·GDP·Mg2+ complexes. Isotope-edited IR studies of the Ras·GTP·Mg2+ complex show that GTP remains bound to Ras at pH as low as 2.0 and that the γ-phosphate is not protonated at pH ≥ 3.3, indicating that the active site decreases the γ-phosphate pKa by at least 1.1 pK a units compared with solution. Amide I studies show that the Ras·GTP·Mg2+ and Ras·GDP·Mg2+ complexes partially unfold in what appear to be two transitions. The first occurs in the pH range 5.4-2.6 and is readily reversible. Differences in the pH-unfolding midpoints for the Ras·GTP·Mg2+ and Ras·GDP·Mg2+ complexes (3.7 and 4.8, respectively) reveal that the enzyme-γ-phosphoryl interactions stabilize the structure. The second transition, pH 2.6-1.7, is not readily reversed. The pH-dependent unfolding of the Ras·GTP·Mg2+ complex provides an alternative interpretation of the data that had been used to support the γ-phosphate mechanism, thereby raising the issue of whether this mechanism is operative in GTPase-catalyzed GTP hydrolysis reactions.

Original languageEnglish (US)
Pages (from-to)9931-9935
Number of pages5
JournalJournal of Biological Chemistry
Volume276
Issue number13
DOIs
StatePublished - Mar 30 2001

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Vibrational spectroscopy
Protonation
Guanosine Triphosphate
Hydrolysis
Spectrum Analysis
Phosphates
GTP Phosphohydrolases
Mutagenesis
Second Messenger Systems
Vibrational spectra
Amides
Isotopes
Protons
Water
Enzymes
Catalytic Domain

ASJC Scopus subject areas

  • Biochemistry

Cite this

γ-phosphate protonation and pH-dependent unfolding of the Ras·GTP·Mg2+ complex : A vibrational spectroscopy study. / Cheng, Hu; Sukal, Sean; Callender, Robert; Leyh, Thomas S.

In: Journal of Biological Chemistry, Vol. 276, No. 13, 30.03.2001, p. 9931-9935.

Research output: Contribution to journalArticle

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